Modulation of inflammation related to columnar epithelia

ABSTRACT

This invention provides pharmaceutical compositions containing lipoxin compounds and therapeutic uses for the compounds in treating or preventing a disease or condition associated with columnar epithelial inflammation. The invention also discloses methods for screening for compounds useful in preventing columnar epithelial inflammation.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. Ser. No. 08/806,278filed Feb. 25, 1997, which is a divisional of U.S. Ser. No. 08/268,049filed Jun. 29, 1994, which is a continuation in part application of U.S.Ser. No. 08/084,311 filed Jun. 29, 1993, which in turn is acontinuation-in-part application of co-pending application U.S. Ser. No.07/748,349, filed Aug. 22, 1991, which in turn is a continuation-in-partapplication of co-pending application U.S. Ser. No. 07/677,388, filedApr. 1, 1991. The contents of the aforementioned applications are herebyincorporated by reference.

GOVERNMENT SUPPORT

[0002] The work leading to this invention was supported by at least onegrant from the U.S. Government. The U.S. Government, therefore, may beentitled to certain rights in the invention.

BACKGROUND

[0003] Columnar epithelia exist in the lungs, kidneys, bladder, bileducts, pancreatic ducts, gall bladder, testicles, thyroid, trachea,intestine, stomach, and liver. In many disease states, polymorphonuclearleukocytes (PMN) migrate across these epithelia. (Yardley J. H., et al.(1977). In The Gastrointestinal Tract. Yardley and B. C. Morson,editors. Williams and Wilkins Co., Baltimore. 57.) (Yardley, J. H.(1986). In Recent Developments in the Therapy of Inflammatory BowelDisease. Proceedings of a Symposium. Myerhoff Center for DigestiveDisease at Johns Hopkins, Baltimore. 3-9.) This migration of PMN is anearly event in the mechanism of epithelial perturbation, which includesone or more of the following events: abnormal fluid and electrolytetransport, specific epithelial barrier dysfunction, and ultimatelymucosal breakdown. These perturbations lead to chronic and episodicinflammatory conditions.

[0004] Epithelial perturbations cause or contribute to many inflammatorydisease states including: gastritis, diverticulitis, cystic fibrosis,infectious colitis, bronchitis, asthma, Crohnis disease, nephritis,alveolitis, intestinal ulcers, idiopathic AIDS enteropathy,gastroenteritis, ischemic diseases, and glomerulonephritis. The efficacyof existing therapy for epithelial inflammation, such as methotrexate orcorticosteroids, is highly unsatisfactory, partially due to a hightoxicity which produces severe, adverse effects such as bone-weakeningand systemic immuno-suppression. (Physician's Desk Reference (41st ed.,1987) Medical Economics Co., Inc. 1103-1104.) Even under idealbioavailability conditions, the existing treatments fail tomechanistically target columnar epithelial inflammation.

[0005] New treatments for epithelial inflammation are needed.

SUMMARY OF INVENTION

[0006] This instant invention discloses new methods and compositions fortreating or preventing inflammation which is caused or contributed to bythe perturbation of columnar epithelia in a subject. The newpharmaceutical compositions comprise natural lipoxin A₄ or analogs oflipoxin A_(4.) And the new methods comprise administering to a subjecthaving a columnar epithelial inflammatory disease an effective,antiinflammatory amount of natural lipoxin A₄ or a lipoxin A₄ analog.

[0007] Natural lipoxin A₄ and analogs are thought to effect-theiranti-inhibitory activity by interfering with the interaction betweenpolymorphonuclear (PMN) cells and columnar epithelium. Migration of PMNis an early event in the mechanism of epithelial perturbation whichleads to mucosal breakdown, epithelial dysfunction, and chronicinflammatory conditions. As disclosed herein, prior exposure ofpolymorphonuclear leukocytes (PMN) to certain lipoxin compounds alterssubsequent PMN migration across the columnar epithelium, therebypreventing an inflammatory response. By inhibiting an early event in themechanism, LXA₄ effectively targets inflammation and inflammatoryresponses caused or contributed to by epithelial perturbation.

[0008] LXA₄, is a naturally-occuring tetraene-containing eicosanoids.Therefore, pharmaceutical compositions of LXA₄ or analogs thereof wouldexpected to be biocompatible. In addition, because LXA₄ and analogsthereof are highly potent in vivo, relatively small doses can beadministered to produce a therapeutic effect. In addition, naturallipoxins are subject to metabolic transformations in situ, that wouldfurther minimize any toxic, adverse effects, or adverse druginteractions. Alternatively, the instant invention discloses LXA₄analogs that are relatively resistant to in vivo degradation andtherefore, if shown to be safe, can be administered for a more prolongedtherapeutic effect. Lipophilic LXA₄ can be actively absorbed by columnarepithelial tissue.

[0009] For the reasons stated above, pharmaceutical compositions ofnatural LXA₄ or analogs thereof provide a superior drug for treatingcolumnar epithelial inflammatory diseases. Additional features andadvantages of the invention will become more apparent from the followingdetailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0010] This invention pertains to methods for treating or preventinginflammation or an inflammatory response caused or contributed to by theperturbation of a columnar epithelium. The term “columnar epithelium” isintended to mean one or more of the epithelia of the intestine, kidney,stomach, liver, thyroid, trachea, lung, gall bladder, urinary bladder,bile ducts, pancreatic ducts, liver, and testicles. A columnarepithelium performs three functions. First, it acts as a physicalbarrier. Second, it moves fluids, electrolytes, and nutrients in vectorsacross the epithelium. Third, it synthesizes and releases bioactivemolecules to influence other cell types.

[0011] An epithelial perturbation is a deleterious alteration of one ormore of the following: the normal barrier function; the transportationof fluids, electrolytes, or nutrients; or the synthesis or release ofbioactive molecules by the epithelial cells. The term “epithelialperturbation” is meant to include one or more of the following events:abnormal fluid and electrolyte transport, especially chloride ionsecretion, specific epithelial barrier dysfunction, and eventual mucosalbreakdown. These perturbations lead to chronic and episodic inflammatoryconditions.

[0012] This invention provides, in part, a method of screening for acompound which attenuates abnormal fluid and electrolyte transportation,which may or may not be caused by activated inflammatory cells. Thisinvention also provides a method of treating or preventing the symptomsof abnormal fluid and electrolyte transportation, such as secretorydiarrhea by administering to a subject of an effective amount of anatural lipoxin or lipoxin analog, or combination thereof, to reduce orprevent an epithelial perturbation of fluid and electrolytetransportation.

[0013] Activation of one or more types of inflammatory cells can mediatethis inflammatory perturbation by inducing inflammatory cell action inthe form of adhesion, migration, the release of bioactive molecules, ora combination thereof. Nonlimiting examples of inflammatory cells areleukocytes, which encompass polymorphonuclear leukocytes (PMN),eosinophils, T-lymphocytes, B-lymphocytes, natural killer cells, andmonocyte/macrophages. For example, migration of PMN across theepithelium of the intestine is an early event in the perturbationmechanism. The term “migration” is meant to include both the adhesion ofPMN to the epithelium and the complete traversion across the epitheliumto the other side. Under normal circumstances, PMN rarely adhere to theepithelial surface, and thus such adhesion is considered therate-limiting step in the migratory process.

[0014] This invention provides, in part, a method of screening for acompound which inhibits the activation of inflammatory cells, such asPMN, which interact with an epithelium. This method evaluates theanti-inflammatory action of an eicosanoid, such as a lipoxin, a lipoxinanalog, or a combination thereof, based on the extent of its inhibitionof PMN migration in the basal-to-apical direction. This invention alsoprovides a method of treating or preventing inflammation or theinflammatory response caused or contributed to by activation ofinflammatory cells. This method is the administration to a subject of aneffective amount of a lipoxin or lipoxin analog, or combination thereof,to reduce or prevent inflammatory cell activation and the consequentinflammatory response.

[0015] This invention is based, in part, upon the finding that priorexposure of PMN to nanomolar concentrations of lipoxin A₄ (LXA₄) andcertain lipoxin analogs modify subsequent PMN migration across anepithelial barrier. The effect was found to be dependent on thedirection of PMN transepithelial migration: LXA₄ inhibited the number ofmigrating PMN cells in the basal-to-apical direction, but promoted thenumber of migrating PMN cells in the apical-to-basal direction. In atypical embodiment of the screening method, the basal-to-apicalinhibition represented a decrease of 25%, and the apical-to-basalpromotion represented an increase of 80%, after pretreatment of PMN withLXA₄ (10 nM) for 15 minutes.

Inflammatory Diseases of Columnar Epithelia

[0016] Epithelial perturbations cause or contribute to inflammatoryintestinal disease states including: acute self-limited enterocolitis;viral infections such as non-specific enteritis or specific viralenteritis; ulcerative colitis; Crohn's disease; diverticulitis;bacterial enterocolitis, such as salmonellosis, shigellosis,campylobacter enterocolitis, or yersinia enterocolitis; protozoaninfections such as amebiasis; helminthic infection; andpseudomembraneous colitis.

[0017] Additional inflammatory intestinal diseases are duodenitisresulting caused by infections, physical and chemical injuries, Celiacdisease, allergic disease, immune disorders or stress ulcers;lymphocytic colitis; collagenous colitis; diversion-related colitis;acute self-limited colitis; microscopic colitis; solitary rectal ulcersyndrome; Behcetts disease; nonspecific ulcers of the colon; secondaryulcers of the colon; ischemic bowel disease; vasculitis; pepticduodenitis; peptic ulcer; bypass enteritis; ulcerative jejunoileitis; ornonspecific ulcers of the small intestine. Malabsorptive disordersinclude mucosal lesions associated with altered immune response such asidiopathic AIDS enteropathy, with viral or bacterial infections, or withmiscellaneous diseases such as mastocytosis or eosinophilicgastroenteritis.

[0018] Perturbations of the epithelia of the lung and trachea cause orcontribute to inflammatory lung diseases such as: cystic fibrosis,bronchiolitis, bronchitis, asthma, interstitial lung disease,eosinophilic pneumonias, tracheobronchitis, tracheoesophageal fistulas,and alveolitis.

[0019] Perturbations of the epithelium of the kidney cause or contributeto diseases such as: glomerulonephritis, nephritis, polycystic disease,ischemic disease, immune-complex-induced disease, immunopathogenicinjuries, pyelonephritis, and tubulointerstitial disease.

[0020] Perturbations of the epithelium of the stomach cause orcontribute to diseases such as gastritis and stomach ulcers.

[0021] This invention also encompasses inflammation of columnarepithelial caused or contributed to by surgery, allergy, chemicalexposure, and physical injury.

Methods of Screening for Anti-inflammatory Compounds

[0022] This invention provides a method of screening for a compoundwhich inhibits activation of inflammatory cells which interact with anepithelium. This method comprises pretreating the inflammatory cell withthe compound, placing the pretreated cell on one side of a preparedepithelial barrier having a chemotactic agent on the other side, anddetermining whether the compound inhibits the activation of theinflammatory cell. Nonlimiting examples of inflammatory cells areleukocytes such as polymorphonuclear leukocytes (PMN), eosinophils,T-lymphocytes, B-lymphocytes, natural killer cells, andmonocyte-macrophages. Inflammatory cell activation includes adhesion tothe epithelium, migration across the epithelium, release of bioactivemolecules, or a combination thereof.

[0023] The epithelial barrier can be constructed by growing epithelialcells and forming a monolayer by controlling the growth media topreserve the polarized phenotype. For example, T84 cells are grown asmonolayers in a 1:1 mixture of Dulbecco-Vogt modified Eagle's medium andHam's F12 medium supplemented with 15 mM Na⁺-HEPES buffer, pH 7.5, 1.2g/l NaHCO₃, 40 mg/l penicillin, 8 mg/l ampicillin, 90 mg/l streptomycin,and 5% newborn bovine serum.

[0024] Normal or inverted monolayers can be constructed using thecommercially available insert system (Costar inserts, 0.33 cm², 5 μmpolycarbonate fibers, Cambridge, Mass.). The larger pore size is crucialto allow inflammatory cells to penetrate the filter. Furthermore, thefilter must be coated with Collagen I to allow epithelial cellattachment. Prepared monolayers should be used within 6-14 days, sincenot only do physiologic responses diminish with time, but also cellprocesses can eventually move through the 5 μm pores and result in adoubled monolayer, with one monolayer on each side of the filter. Themonolayer may be inverted or not, to allow screening for migration,adhesion, or release of bioactive molecules in both the apical-to-basaldirection and the basal-to-apical direction.

[0025] Nonlimiting examples of cells from which to form the epithelialbarrier include: the intestinal cell lines Caco-2 (ATCC accession numberHTB 37), IEC-6 (ATCC accession number CRL 1592), T84 (ATCC accessionnumber CCL 248) or HT-29 (ATCC accession number HTB 38); the renaltubular cell lines MDCK (ATCC accession numbers CCL 34 and CRL 6253) orLLC-PK₁ (ATCC accession numbers CL 101 and CRL 1392); and isolatedalveolar epithelial cells grown in primary culture.

[0026] The prepared epithelial barrier may optionally have a permeableartificial membrane on one side to prevent membrane-membrane contactbetween the epithelial barrier and the inflammatory cell. While thereare numerous artifical supports available, a preferable membrane made ofpolycarbonate may be obtained commercially from Costar Corp., Cambridge,Mass.

[0027] The epithelial barrier also may have cell-sized objects(approximately 7-10 μm in diameter) located in the interstitial spacesbetween the epithelial barrier cells. These objects can be actual cells,or latex beads. The latex beads can be inert or coated with one or moretypes of active molecules attached to the bead surface, such as markermolecules, signal molecules, or monoclonal antibodies. The inert beadsare available commercially (Seradyne, Indianapolis, Ind.). The beadsmimic the physical presence of inflammatory cells. In addition, thecoated beads provide a high local concentration of the coatingmolecule(s) and minlic the structural stability of cell-cell membranecontact. Furthermore, the beads provide a method of introducingbioactive molecules of otherwise low solubility into the system for longperiods of time. The beads may be coated with a particular selectedmolecule, without undue experimentation, by methods known to thoseskilled in the art.

[0028] A chemotactic agent elicits the adhesion, migration, release of abioactive molecule, or combination thereof by the inflammatory cells onthe opposite side of the epithelial barrier. Nonlimiting examples of anappropriate chemotactic agent are: eicosanoids such as leukotriene B₄(LTB₄), 12S-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (12-HETE),and 5S-hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid (5-HETE); IL-8,IGF-β, C5a, platelet activating factor (PAF), and N-formyl-Met-Leu-Phe(fALP). In addition, any microbial pathogen-derived chemotactic factormay be used, since fMLP is a model attractant for bacterial chemotaxis.The amount of chemoattractant should be sufficient to elicit adhesion,migration, or release of a bioactive molecule in the absence of aninhibiting compound for the particular barrier system being used. Forexample, a concentration of 1 μM fMLP may be used.

[0029] Measuring the inhibition of inflammatory cell activation can beachieved in several ways. The relative number of migrating PMN cells canbe measured, for example, by a myeloperoxidase assay. The effect of cellactivation, in the form of specific barrier dysfunction or abnormalelectrolyte transport, can also be evaluated with electrophysiologicalmeasurements of the electrical resistance of the epithelial barrier, theelectrical resistance of the epithelial cell membrane, and/or theendogenous cell current.

[0030] In a typical embodiment, the method would be a method ofscreening for a compound which modifies PMN adhesion to or migrationacross an intestinal epithelial barrier. This method comprisespretreating PMN with the compound, placing the pretreated PMN on oneside of the intestinal barrier having a chemotactic agent on the otherside, and determining whether the compound modifies PMN adhesion to ormigration across the barrier. The epithelial barrier may be modeled bycolumnar epithelial cells with features similar to those of naturalcrypt epithelial, such as but not limited to a monolayer of humanintestinal epithelial cell line T84. The chemotactic agent is fMLP (1μM). The determination of the effectiveness of the compound is measuredby the relative change in migration or adhesion of the PMN as measuredby a myeloperoxidase assay. ( Madara, J. L. et al. (1992) J. Tiss. Cult.Meth. 14:209-216.) Experimental details of this embodiment of thescreening method are provided in Example 1 below.

[0031] This invention also provides a method of screening for a compoundwhich attentuates the effect of an activated inflammatory cell upon acolumnar epithelium, thereby attenuating one or more deleteriousperturbations. This method comprises: combining an inflammatory cellwith a prepared epithelial barrier, pretreating this combination withthe compound, adding an activating agent, and determining whether thedeleterious perturbations are attentuated by the compound.

[0032] The activating agent is an agent which stimulates the activationof the the inflammatory cell. Nonlimiting examples of an inflammatorycell activating agent are: phorbol ester, a Ca⁺² ionophore,phytohemaglutinin, chemotactic agents as described above, and endotoxin.In addition, the activating agent may have an effect on both theinflammatory cell and the epithelial cell. Nonlimiting examples of thesekinds of activating agents are cytokines such as γ-IFN. The preparedepithelial barrier can be made as described above.

[0033] The attenuation can be measured in terms of electrical parameterssuch as the electrical resistance of the epithelial barrier, theelectrical resistance of the epithelial cell membrane, or the endogenouscurrent, or combinations thereof. The relative attenuation is thecomparison of electrical parameters in the presence and absence of thecompound.

[0034] A typical embodiment of this method will be used to screen for acompound which reduces or eliminates the symptoms of secretory diarrheacaused by abnormal chloride secretion. The PMN-derived paracrine factorthat elicits chloride secretion from T84 intestinal epithelial cellmonolayers is 5′-adenosine monophosphate (5′-AMP). (Madara, J. L. et al.(1993) J.Clin. Invest. 91: 2320-2325.) The method comprises: combiningan intestinal epithelial barrier with PMN cells, stimulating chloridesecretion by an intestinal epithelial cells with an amount of 5′-AMP oran agonist thereof, exposing the epithelial cells to the compound; anddetermining the attenuating effect of the compound upon the activationof the epithelial cells. The attenuation is measured by the electricalresistance of the epithelial barrier, the electrical resistance of theepithelial cell membrane, and/or the endogenous cell current.

[0035] Nonlimiting examples of 5′-AMP agonists are cyclic AMP,forskolin, and carbachol. Nonlimiting examples of variable rangesappropriate for a standard dose-response curve are: 5′-AMP (10⁻⁸-10⁻³ M,in the apical direction; 10⁻⁷-10⁻² M in the basal direction); cAMP andforskolin (10⁻⁸-10⁻² M); and carbachol (10⁻⁸-10⁻³ M). For example,incremental steps of one-half log concentrations may be used. The amountof the 5′-AMP or agonist should be an amount sufficient to elicitintestinal chloride secretion. The following Example 2 discloses theexperimental details for performing the electrophysiologicalmeasurements.

[0036] The intestinal epithelial barrier may be from, but is not limitedto, any of the above mentioned intestinal cell lines, especially the T84cell line. In addition, the screened compound may be, for example, aneicosanoid such as a lipoxin or lipoxin analog. The lipoxin analog mayhave a longer tissue half-life than the corresponding lipoxin, or may beactively absorbed by the intestine, or both.

Lipoxins, Lipoxin Analogs, and Combinations Thereof

[0037] Lipoxin compounds (e.g. natural lipoxins and lipoxin analogs) canbe administered to a subject for the treatment or prevention ofinflammation or inflammatory responses caused or contributed to byepithelial perturbations. Preferred lipoxin compounds are naturallipoxin A4 (LXA₄) and analogs thereof.

[0038] “Natural lipoxins” are lipoxygenase-derived, biologically activeeicosanoids produced by PMN, platelets, eosinophils and macrophages.(Samuelsson B., et al. (1987). Science 237:1171-1176); (Dahlen S. E.,and C. N. Serhan (1991). In Lipoxygenases and Their Products, AcademicPress. New York, N.Y. 235-276). These compounds have been shown toelicit selective counterregulatory responses in human PMN in vitro,including the inhibition of leukotriene B₄ (LTB₄) and fMLP-stimulatedchemotactic responses across Boyden chambers (filters) (Lee T. H., etal. (1989). Clin Sci. 77:195-203); (Lee T. H., et al. (1991). BiochemBiophys Res Comm 180:1416-1421), blocking of Ca²⁺ mobilization (SpringerT. A . (1990). Nature 346:196-197), and inhibition of LTD₄-inducedadhesion to mesangial cells (Brady H. R., et al. (1990). Am J. PhysioL259 (Renal Fluid Electrolyte Physiol. 28): F809-815). In vivo, lipoxinsare potent inflammatory mediators which act to inhibit lymphocytemigration across vascular endothelia (Hedqvist P. J. et al. (1989).Acta. Physiol Scand. 137: 571-572), decrease LTD₄-inducedvasoconstriction (Badr K. F., et al. (1989). Proc. Nat Acad. Sct U.S.A.86:3486-3442), and modulate LTD₄-induced airway obstruction, (ChristieP. E., et al. (1992) Am Rev RespirDis 145:1281-1284). Lipoxins includethe bioactive (5S, 14R,15S)-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid (LXB₄), andmore preferably,(5S, 6R, 15S)-trihydroxy-7,9,13-trans-I1-cis-eicosatetraenoic acid (LXA₄).

[0039] In addition to natural lipoxins, lipoxin analogs are usefulantiinflammatory agents. “Lipoxin analogs” include compounds which arestructurally similar to natural lipoxins, compounds which share the samereceptor recognition site, compounds which share the same or similarlipoxin metabolic transformation region as lipoxin, and compounds whichare art-recognized as being analogs of lipoxin. Lipoxin analogs alsoinclude metabolites of lipoxin and lipoxin analogs. A nonlimitingexample of a lipoxin analog which inhibits PMN migration across anepithelial barrier is 11-trans-LXA₄. (See Example 1). Some lipoxinanalogs are sufficiently lipophilic to be actively absorbed by theintestine. Generally, lipophilic analogs will have relatively short(C₂-C₄) hydrocarbon groups occupying the C-16+ position, as in thenatural lipoxin compound.

[0040] One particularly suitable class of lipoxin analogs for use in theinstant invention are those exhibiting a longer tissue half-life thancorresponding natural lipoxins. A “lipoxin analog having a longer tissuehalf-life than corresponding lipoxins” refers to a compound which has an“active region” that functions like the active region of a naturallipoxin (e.g. LXA₄ or LXB₄), but which has a “metabolic transformationregion” that differs from natural lipoxin. By“active region” is meantthe region of a natural lipoxin or lipoxin analog, which is associatedwith in vivo cellular interactions. The active region may bind the“recognition site” of a cellular lipoxin receptor or a macromolecule orcomplex of macromolecules, including an enzyme and its cofactor.Preferred lipoxin A₄ analogs have an active region comprising C5-C15 ofnatural lipoxin A₄. Preferred lipoxin B₄ analogs have an active regioncomprising C5-C14 of natural lipoxin B4.

[0041] The term “metabolic transformation region” refers to that portionof a lipoxin, a lipoxin metabolite, or lipoxin analog including alipoxin analog metabolite, upon which an enzyme or an enzyme and itscofactor attempts to perform one or more metabolic transformations whichthat enzyme or enzyme and cofactor normally transform on lipoxins. Themetabolic transformation region may or may not be susceptible to thetransformation. A nonlimiting example of a metabolic transformationregion of a lipoxin is a portion of LXA₄ that includes the C-13,14double bond or the C-15 hydroxyl group, or both.

[0042] The pathway of lipoxin metabolism includes dehydrogenation,reduction of at least one unsaturated carbon-carbon bond, and/orω-oxidation. These enzymatic transformation occur within the C-12 toC-20 portion of an LXA₄ analog, for example. Therefore, a lipoxin analogwith a longer tissue half-life may be designed with chemicalmodifications which inhibit, resist, or raise the transition stateenergy of an analog or its metabolite for at least one of the metabolictransformations. Such analogs employ electronic effects at the relevantcarbon atom, steric effects, and/or potential suicide substrate moietiessuch as those that allow covalent Michael addition to a metabolicenzyme.

[0043] Nonlimiting examples of a LXA₄ analog having a longer tissuehalf-life than LXA₄ include LXA₄ analogs with C-15 and/or C-16substitutions such as: mono- or di-hydroxyl, methyl, fluoromethyl, andfluoro; C-16 substitutions such as phenyl, halo-substituted phenyl, andalkoxy; and C-19 or C-20 substitutions such as fluoromethyl, phenyl, andfluoro; and 13-yne or 14-yne substitutions. It is known that theintestine actively absorbs lipophilic fatty acids, especially those twoto four carbon atoms in length. (Binder, H. J. In, GastrointestinalDisease, 4th ed. (Sleisenger, M. H., and Fordtran, J. S., eds.) W. B.Saunders Co., Phildelphia, 1989, pp. 1022-1045.) In other emgodiments,similar substitutions create structural analogs based on other lipoxinssuch as LXB₄.

[0044] In the most preferred embodiment of this invention, the compoundsof this invention have the following structural formulas:

[0045] where R′ is H or CH ₃.

[0046] In other preferred embodiments of this invention, the compoundsof this invention have the following structural formulas:

[0047] This invention also contemplates use of combinations of lipoxinsand lipoxin analogs. A nonlimiting example of a combination is a mixturecomprising a lipoxin analog x which inhibits one enzyme whichmetabolizes lipoxins and which optionally has specific activity with alipoxin receptor recognition site, and a second lipoxin analog y whichhas specific activity with a lipoxin receptor recognition site and whichoptionally inhibits or resists lipoxin metabolism. This combinationresults in a longer tissue half-life for at leasty since x inhibits oneof the enzymes which metabolize lipoxins. Thus, the lipoxin actionmediated or antagonized byy is enhanced.

Methods of Making Lipoxins and Lipoxin Analogs

[0048] Lipoxins may be isolated as described (Serhan, C. N. et al.(1990) Methods in Enzymol. 187:167) from biological sources, synthesizedor obtained commercially. LXA₄ and LXB₄ are available from Biolmol, Inc.(Philadelphia, Pa.) and Cayman Biochemical (Ann Arbor, Mich.). LXA₄,LXB₄, and the 11-trans-LXA₄ isomer are available from CascadeBiochemical, Ltd (Berkshire, UK). Nonlimiting examples of the structuresand syntheses of both lipoxins and lipoxin analogs, including methylesters of lipoxin analogs, are illustrated in the following patents andpublications:

[0049] (1) Nicolaou, K. C. et al. Identification of a novel7-cis-11-trans-LXA₄ generated by human neutrophils: total synthesis,spasmogenic activities and comparison with other geometric isomers oflipoxins A₄ and B₄ (1989). Biochim. Biophys. Acta 1003:44-53;

[0050] (2) Nicolaou, K. C. et al. Total synthesis of novel geometricisomers of LXA₄ and LXB₄ (1989). J. Org. Chem. 54: 5527-5535;

[0051] (3) Nicolaou, K. C. et al. Lipoxins and related eicosanoids:biosynthesis, biological properties, and chemical synthesis (1991).Angew. Chem Int Ed. Engl 30: 1100-1116;

[0052] (4) U.S. Pat. Nos. 4,576,758; 4,560,514; 5,079261; and 5,049,681;and

[0053] (5) JP Patent Nos. 3,227,922; 63,088,153; 62,198,677; and1,228,994.

[0054] Preferred lipoxin analogues having a longer half-life thannatural lipoxins can be prepared as described in the following Example 2

Methods of Treatment

[0055] This invention provides, in part, method of treating orpreventing inflammation or an inflammatory response caused orcontributed to by the activation of inflammatory cells which interactwith a columnar epithelium. The interaction between activatedinflammatory cells and the epithelium results in one or more epithelialperturbations. This anti-inflammatory treatment is the administration toa subject of an effective amount of a lipoxin, lipoxin analog, orcombination thereof to inhibit the activation of the inflammatory cellsuch that the epithelial perturbation and inflammation or aninflammatory response are significantly reduce4 or eliminated.

[0056] A significant reduction of inflammation or an inflammatoryresponse includes reducing or eliminating one or more of the symptomsassociated with inflammation. For example, PMN transmigration stimulateselectrogenic chloride secretion, which is the basis of secretorydiarrhea, one of the symptoms of inflammatory bowel diseases. (ash, S.et al. (1991). J.Clin. Invest. 87:1474-1477.) Additional nonlimitingexamples of symptoms of inflammatory bowel diseases are crampingabdominal pain, malabsorption, dehydration, bloody stool, or fever. Inaddition to the inflammatory bowel disease listed above, bowelinflammation may also result from surgery, allergy, chemical exposure,or physical injury. Reduction of epithelial perturbation can alsoinclude inhibition of inflammatory cell activation. For example, areduced perturbation can be the inhibition of PMN migration in thebasal-to-apical direction represented by a decrease of at least about25%.

[0057] Lipoxins include LXA₄ or LXB₄. The lipoxin analog can have alonger tissue half-life than the corresponding natural lipoxin. Thelipoxin analog can also be lipophilic. The lipoxin analog can also beactively absorbed by the intestine. Lipoxins, lipoxin analogs, andcombinations of lipoxins as used in these methods of treatment aredefined above in the preceding two sections.

[0058] This invention also provides a method for the treatment orprevention of one or more of the symptoms of inflammatory diseases ofcolumnar epithelia. In this method, the epithelial perturbations whichcause or contribute to these symptoms may or may not be mediated byinflammatory cells. This method of treatment comprises theadministration to a subject of an effective amount of a lipoxin, lipoxinanalog, or combination thereof such that the epithelial inflammation orinflammatory response is significantly reduced or eliminated.

[0059] A significant reduction of inflammation or an inflammatoryresponse includes reducing or eliminating one or more of the symptomsassociated with inflammation. For example, abnormal chloride secretioncauses or contributes to secretory diarrhea, a symptom of inflammatorybowel diseases. 5′AMP elicits chloride secretion from T84 intestinalepithelial cell monolayers, iixa manner which may not always bedependent upon PMN. (Nadara, J. L. et al. (1993) J.Clin. Invest.91:2320-2325.) Additional nonlimiting examples of symptoms ofinflammatory bowel diseases are cramping abdominal pain, malabsorption,dehydration, bloody stool, or fever.

[0060] Lipoxins include LXA₄ or LXB₄. The lipoxin analog may havecharacteristics such as a longer tissue half-life than the correspondingnatural lipoxin, be lipophilic, or be actively absorbed by theintestine, or a combination thereof. Lipoxins, lipoxin analogs, andcombinations of lipoxins as used in these methods of treatment aredefined above.

[0061] In one embodiment, the lipoxin or lipoxin analog independentlyacts to modulate epithelial perturbations, such as chloride ionsecretion. Without intending to be bound, it is speculated that lipoxinsand lipoxin analogs, independent of PMN activation, can decreasechloride ion secretion to an extent that secretory diarrhea issignificantly reduced.

Pharmaceutical Compositions and Packaged Drugs

[0062] This invention also encompasses pharmaceutical compositions andpackaged drugs containing lipoxins, lipoxin analogs, salts thereof, andcombinations thereof for the treatment of inflammation and inflammatoryresponses in a subject. In one embodiment of this invention, thepharmaceutical compositions and packaged drugs are for the treatment orprevention of the columnar epithelial perturbations related to PMNactivation in inflammatory bowel diseases.

[0063] The term “subject” is intended to include living organismssusceptible to conditions or diseases caused or contributed to byinflammation and inflammatory responses. Examples of subjects includehumans, dogs, cats, cows, goats, and mice. The term “subject” is furtherintended to include transgenic species,

[0064] The term “pharmaceutically acceptable salt” is intended toinclude art-recognized pharmaceutically acceptable salts. Thesenon-toxic salts are usually hydrolyzed under physiological conditions,and include organic and inorganic bases. Examples of salts includesodium, potassium, calcium, ammonium, copper, and aluminum as well asprimary, secondary, and tertiary amines, basic ion exchange resins,purines, piperazine, and the like. The term is further intended toinclude esters of lower hydrocarbon groups, such as methyl, ethyl, andpropyl. In this paragraph, the next paragraph, and in the discussion ofmethods of treatment and pharmaceutical compositions, it should beunderstood that references to lipoxin analogs are meant to includecorresponding pharmaceutically acceptable salts.

[0065] The term “pharmaceutical composition” comprises one or morenatural Ilipoxin or lipoxin analog as an active ingredient(s), or apharmaceutically acceptable salt(s) thereof, and may also contain apharmaceutically acceptable carrier and optionally other therapeuticingredients. The compositions include compositions suitable for oral,rectal, ophthalmic, pulmonary, nasal, dermal, topical, parenteral(including subcutaneous, intramuscular and intravenous) or inhalationmodes of administration. The most suitable route in any particular casewill depend on the nature and severity of the conditions being treatedand the nature of the active ingredient(s). The compositions may bepresented in unit dosage form and prepared by any of the well-knownmethods.

[0066] Appropriate dosage regimes for treating a particular disease orcondition associated with columnar epithelial inflammation can bedetermined empirically by one of skill in the art and may be adjustedfor the purpose of improving the therapeutic response. For example,several divided dosages may be administered daily or the dose may beproportionally reduced over time. A person skilled in the art normallymay determine the effective dosage amount and the appropriate regime. Aless potent lipoxin analog composition may be selected to treat mild orhighly localized inflammation, while a larger dosage or more potentlipoxin analog may be selected to treat severe or widespreadinflammatory episodes. An “effective anti-inflammatory amount” of alipoxin containing pharmaceutical composition for treating a disease orcondition associated with a columnar epithelial inflammation shall meanthat amount that ameliorates the inflammation and eliminates the syptomsof the disease. An “effective anti-diuretic amount” of a lipoxincontaining pharmaceutical composition is that amount that restorestransportation of fluid, electrolytes, or nutrients by a columnarepithelium to the normal, homeostatic level.

[0067] The term “packaged drug” is meant to include one or more dosagesof an effective pharmaceutical composition of a lipoxin, a lipoxinanalog, salt thereof or combination thereof, a container holding thedosage(s), and instructions for administering the dosage(s) to a subjectfor treatment or prevention of inflammation or an inflammatory response.

[0068] The present invention is further illustrated by the followingexample which should in no way be construed as being further limiting.The contents of all references and issued patents cited throughout allportions of this application including the background are expresslyincorporated by reference.

Example 1 Lipoxin A₄ Modulates Migration of Human PMNs Across Intestinal

[0069] Epithelial Monolayers.

[0070] Lipoxins Synthetic LXA₄, LXB₄, and 11-trans-LXA₄ were obtainedfrom Cascade Biochem Ltd. (Berkshire, United Kingdom). Concentrationswere determined from extinction coefficients as described in SheppardK-A., et al. (1992). Biochimica et Biophysica Acta 1133:223-234. Alleicosanoid stock solutions were stored at −70° C. in methanol (AmericanScientific Products). Eicosanoids were diluted in modified HBSS to aconcentration of 1 μM prior to all experiments. PMN or T84 monolayerswere exposed to lipoxins at indicated concentrations and allowed toincubate at 37° C. for the indicated period of time. Vehicle controlsconsisted of dilutions of the solvent (ethanol)-equivalent to thehighest concentration of lipoxin used in any given experiment (0.01%).

[0071] Cell culture Approximately 350 epithelial monolayers were usedfor these studies. T84 intestinal epithelial cells (passages 70-95) weregrown and maintained as confluent monolayers on collagen coatedpermeable support. Monolayers were grown on 0.33 cm² ring-supportedpolycarbonate filters (Costar Corp., Cambridge, Mass.) and utilized 6-14days after plating as described in Example 2. Transepithelial resistanceto passive ion flow was measured as described in Parkos C. A., et al.(1991). J. Clin. Invest. 88:1605-1612); and Parkos C. A., et al. (1992).J. Cell. Biol. 117:757-764). Inverted monolayers used to study migrationof PMN in the basolateral-to-apical direction were constructed asdescribed in Parkos C. A., et al. (1991). J. Clin. Invest.88:1605-1612).

[0072] Migration assay: The PMN transepithelial migration assay has beendetailed in Nash S., etal. (1987). J. Clin. Invest. 80:1104-1113; NashS., et al. (1991). J. Clin. Invest. 87:1474-1477); Parkos C. A., et al.(1991). J. Clin. Invest. 88:1605-1612); and Parkos C. A., et al. (1992).J. Cell. Biol. 117: 757-764. Briefly, human PMN were isolated fromnormal human volunteers and suspended in modified HBSS (without Ca²⁺ andMg²⁺, with 10 mM Hepes, pH 7.4, Sigma) at a concentration of5×10^(7/)ml. Prior to addition of PMN, T₈₄ monolayers were extensivelyrinsed in HBSS to remove residual serum components. Migration assayswere performed by the addition of PMN (40 μl) to HBSS (160 μl) in theupper chambers after chemoattractant (1 μM fMLP in HBSS) was added tothe opposing (lower) chambers. Unless otherwise indicated, PMN were notwashed free of LXA₄ prior to addition to monolayers, and therefore, afive-fold dilution of lipoxin was present during the migration assay.For apical-to-basolateral migration experiments, PMN (2×10⁶) were addedat time 0. Migration in the basolateral-to-apical direction, whilequalitatively similar, is substantially more efficient than in theapical-to-basolateral direction. (Parkos C. A., et al. (1991). J. Clin.Invest. 88:1605-1612). Therefore, 5-fold fewer PMN (4×10⁵) were addedwhen migration proceeded in the basolateral-to-apical direction in orderthat baseline migration signals be approximately equivalent in bothdirections. (Parkos C. A., et al. (1991). J. Clin. Invest. 88:1605-1612). Migration was allowed to proceed for 120 minutes, unlessotherwise noted. All PMN transepithelial migration experiments wereperformed in a 37° C. room to ensure that epithelial monolayers,solutions, plasticware, etc., were maintained at uniform 37° C.temperature.

[0073] When used, inhibitors to cyclooxygenase (indomethacin, Sigma),leukotriene biosynthesis (MK886, a kind gift from Merck Frosst),G-proteins (pertussis toxin, Calbiochem), or protein kinase C (H7,Sigma; staurosporine, Sigma) were pre-incubated with PMN at indicatedconcentrations for 15 min at 37° C. Inhibitors were washed free from PMNby two washes with HBSS. PMN were subsequently exposured to LXA₄ (10 nM)and PMN transepithelial migration was assessed as described above in theapical-to-basolateral direction.

[0074] Migration was quantitated by assaying for the PMN azurophilicgranule marker myeloperoxidase (MPO) as described previously (Parkos C.A., et al. (1991). J. Clin. Invest. 88:1605-1612). Following eachmigration assay, non-adherent PMN were extensively washed from thesurface of the monolayer and PMN cell equivalents (PMN CE), estimatedfrom a standard curve, were assessed as the number of PMN's associatedwith the monolayer, the number which had completely traversed themonolayer (ie. across the monolayer into the reservoir bath), as well asthe total number of transmigrating PMN (the sum of monolayer andreservoir-associated PMN).

[0075] Data Presentation: Individual experiments were performed usinglarge numbers of uniform groups of monolayers and PMN from individualblood donors on individual days. PMN isolation was restricted to fivedifferent blood donors (repetitive donations) over the course of thesestudies. Myeloperoxidase assay data were compared by two-factor analysisof variance (ANOVA) or by comparison of means using Student's t-Test.PMN migration results are represented as PMN CE derived from a dailystandard PMN dilution curve. Monolayer-associated PMN are represented asthe number of PMN CE per monolayer and reservoir-associated PMN (ie. PMNwhich had completely traversed the monolayer into the lower chamber) arerepresented as the number of PMN CE/ml (total volume of 1 ml). Valuesare expressed as the mean and s.e.m. of n experimnents.

[0076] Results

LXA₄ Exosure to T84 Epithelial Monolayers Does Not Alter SubsequentfMLP-induced PMN Migration

[0077] PMN can be induced to transmigrate across T₈₄ epithelialmonolayers in response to a transepithelial gradient of the chemotacticpeptide fMLP (1 μM).(Nash S., et al. (1987). J. Clin. Invest. 80:1104-1113); (Parkos C. A., et al. (1991). J. Clin. Invest 88:1605-1612). To determine whether LXA₄ exposure to T₈₄ intestinalepithelial cells influenced subsequent PMN migration, epithelial cellmonolayers were incubated with LXA₄ at a concentration 10 nM for 15 minat 37° C. (conditions which elicit significant effects when PMN's arepre-exposed to LXA₄, see below), with and without removal of LXA₄ frommonolayers, followed by addition of untreated PMNIs under chemotacticconditions. In these experiments, PMN migration across T84 monolayersexposed to LXA₄ did not differ from vehicle control (14.8±1.4 vs15.7±1.8×10⁴ PMN CE/ml for control and LXA₄ exposed monolayers, n=6each, n.s.). Removal of LXA₄ from monolayers by washing thrice with HBSSprior to addition of PMN had no apparent effect on the total number oftransmigrating PMN (14.3±1.1×10⁴ PMN CE/ml, n=6, n.s. compared to eithercontrol or LXA₄ exposed monolayers).

[0078] In addition, exposure of intestinal epithelial cells to LXA₄ didnot significantly influence the integrity of T84 epithelial monolayers.To examine this, transepithelial resistance to passive ion flow wasassessed prior to, and after addition of 10 nM LXA₄ to T84 intestinalepithelial monolayers for 2 hrs (simulated conditions for entiremigration assay period). During this period, transepithelial resistancedid not significantly decrease following addition of LXA₄ (baselineresistance 1255±56 ohm-cm² and 1089±108 ohm-cm² after 2 hr, n=8, n.s.).These results suggest that monolayer integrity, as assessed bytransepithelial resistance, was not affected by LXA₄ treatment and thatepithelial pre-exposure to LXA₄ has no subsequent effect on fMLP-inducedPMN migration.

LXA₄ Does Not Stimulate Migration

[0079] To investigate whether LXA₄ could serve to stimulate PMNmigration in this assay system, dilutions of LXA₄ in the range of0.01-10 nM were placed in the lower chamber of migration wells.Untreated PMN's were added to the upper chamber and assessed forchemotactic capacity toward LXA₄ in the apical-to-basolateral direction.LXA₄ was no more effective than HBSS in promoting PMN migration;compared to FMLP (1 μM), PMN migration toward LXA₄ resulted in a totalof 6±2.1, 9±3.6, 6±2.4, and 12±1.4% of fMLP-induced PMN migration for0.01, 0.1, 1.0, and 10 nM LXA₄, respectively. In the absence of achemotactic gradient (HBSS), 10±2.9% of fMLP-induced migration occurred.These results indicate that LXA₄, in the concentrations tested, did notstimulate PMN transepithelial migration.

Pre-exposure of PMN to LXA₄ Enhances fMLP-induced PMN Migration In theApical-to-basolateral Direction

[0080] To determine if PMN exposure to LXA₄ alters subsequentIMLP-induced migration, PMN were incubated 10 nM LXA₄ for 15 minutes,then added directly to the apical surface of T84 epithelial monolayers,and subsequently assessed for their ability to traverse T84 epithelialmonolayers using a myeloperoxidase assay. (Parkos C. A., et al. (1991).J. Clin. Invest. 88: 1605-1612). PMN (5×10^(7/)ml) were pre-incubatedwith 10 nM LXA₄ for 15 min. at 37° C. and layered on the apical surfaceof washed T84 epithelial monolayers at a density of 2×10⁶lmonolayer. PMNwere driven to transmigrate basolaterally under the influence of a 1 μMgradient of fMLP.

[0081] Results were obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) from washed monolayers, lower reservoirs and totalMPO activity after 120 min, relative to a known standard number of PM.Since tight junctions are the rate limiting barrier to passiveparacellular permeation, transmigration is defined as movement of PMNacross the tight junction. Since monolayer-associated PMN were largelybelow the tight junction (see results), total transmigration in theapical-to-basolateral direction equals the sum of PMN in the oppositereservoir plus monolayer PMN. Data are pooled from 9 individualmonolayers in each condition and results are expressed as the mean andSEM.

[0082] PMN pre-exposure to LXA₄ resulted in significantly increased PMNtransepithelial migration in the apical-to-basolateral direction.Increased PMN migration was evident in both monolayer-associated PMNnumbers (2.98±0.57 vs. 6.93±1.77×10⁴ PMN CE/monolayer for vehiclecontrol and LXA₄ exposed PMN, respectively, p<0.001), as well as thenumber of PMN which completely traversed the epithelial monolayer(6.61±0.50 vs. 11.02±2.91×10⁴ PMN CE/ml for vehicle control and LXA₄exposed, respectively, p<0.01), resulting in a nearly 2-fold increase inthe total number of transmigrating PMN (9.58±1.05×10⁴ PMN CE/ml forvehicle control and 17.95±2.15×10⁴ PMN CE/ml for LXA₄ pre-exposed PMN,p<0.01). As reported previously, (Parkos C. A., et al. (1991). J. Clin.Invest. 88: 1605-1612), examination of 1 μm T84 epithelial monolayersections have revealed that PMN are only rarely associated with theapical epithelial surface and the majority of monolayer associated PMNare found subjunctionally, indicative of migration. Therefore,monolayer-associated PMN in this apical-to-basolateral assay areconsidered transmigrated across the tight junction, the rate limitingbarrier in PMN transepithelial migration. (Parkos C. A., et al. (1991).J. Clin. Invest. 88:1605-1612).

[0083] To further characterize this transmigratory event, PMN werepre-exposed to LXA₄ (10 nM) for various periods of time and subsequentlyassessed for their ability to transmigrate across T84 epithelialmonolayers in the apical-to-basolateral direction. PMN (5×10⁷/ml) werepre-incubated with 10 nM LXA₄ for various periods of time in the rangeof 0-60 min at 37° C. or pre-incubated with various indicatedconcentrations of LXA₄ for 15 min. at 37° C. and layered on the apicalsurface of washed T84 epithelial monolayers at a density of2×10⁶/monolayer. PMN were driven to transmigrate baolaterally under theinfluence of a 1 μM gradient of fMLP. Results were again obtained byharvesting the PMN specific enzyme myeloperoxidase (MPO) from washedmonolayers, lower reservoirs and total MPO activity after 120 min,relative to a known standard number of PMN. Data are pooled from 7-10individual monolayers in each condition and results are expressed as themean and SEM.

[0084] Pre-exposure of PMN to LXA₄ resulted in increased total PMNmigration after a LXA₄ pre-exposure period of 5-30 min. (compared tovehicle controls, for PMN's pre-exposed to 10 nM LXA₄, migrationincreased by 50, 68 and 51% at 5, 15 and 30 min. exposure times,respectively, all p<0.025). Migration had returned to vehicle controlvalues by 45 and 60 minutes of PMN pre-exposure to LXA₄. Pre-exposure ofPMN to LXA₄ was found to be a necessary prerequisite for LXA₄ action onstimulating PMN migration. Indeed, exposure of PMN's to 10 nM LXA₄immediately prior to their addition to epithelial monolayers (ie. 0 min.pre-exposure) resulted in no effect on subsequent fP-induced PMNmigration ( 16.34±4.07 vs. 16.61±3.56×10⁴ total PMN CE/ml for vehiclecontrol and LXA₄ pre-exposure for 0 min., respectively, n.s.). The LXA₄pre-exposure time-dependent enhancement of subsequent neutrophilmigration was largely due to reservoir-associated PMN (11.06±3.05 vs18.02±3.35, 19.96±3.18, 19.64±3.54×10⁴ PMN CE/ml for vehicle control andPMN LXA₄ pre-exposure times of 5, 15 and 30 minutes, respectively,two-factor ANOVA p<0.01). However, a significant increase in the numberof monolayer-associated PMN occurred at 15 minutes of LXA₄ pre-exposure(4.17±1.17 for vehicle control vs 7.55±0.71×10⁴ PMN CE/monolayer forPMN's exposed to 10 nM LXA₄, p<0.01).

[0085] The effect of LXA₄ pre-exposure to PMN and subsequent PMNtransepithelial migration in the apical-to-basolateral direction wasfound to be concentration dependent. Pre-exposure of PMN to LXA₄concentrations in the range of 1.0 pM-10 nM for 15 minutes at 37° C.elicited increased PMN migration at doses of 0.1, 1.0 and 10 nM fmalconcentrations. Similar to the time-course data presented above,LXA₄-elicited stimulation of PMN migration was manifest as an increasein the number of PMN in lower reservoirs (11.06±3.05 vs 20.38±4.83,19.96±4.83, 15.43±4.65, 14.11±3.01, and 13.71±4.14×10⁴ PMN CE/ml forvehicle control and PMN LXA₄ pre-exposure doses of 10, 1, 0.1, 0.01, and0.001 nM, respectively, for 15 min., 37° C., two-factor ANOVA, p<0.025).

[0086] To determine whether the stimulatory action of LXA₄ was presentthroughout the incubation period, PMN's were pre-exposed to LXA₄ (10 nM,15 min), layered on the apical surface of T84 monolayers and driven totransmigrate basolaterally. Monolayers were harvested at various timepoints during migration and assayed for PMN by myeloperoxidase content.PMN (5×10^(7/)ml) were pre-incubated with 10 nM LXA₄ for 15 min. at 37°C. and layered on the apical surface of washed T84 epithelial monolayersat a density of 2×10^(6/)monolayer. PMN were driven to transmigratebasolaterally under the influence of a 1 μM gradient of fMLP.

[0087] Results were obtained by assaying the PMN specific enzymemyeloperoxidase (MPO), relative to a known standard number of PMN. TotalMPO activity (including reservoir- and monolayer-associated MPOactivity) was tested. Data were pooled from 6 individual monolayers ineach condition and results were expressed as the mean and SEM.

[0088] The stimulatory effect of LXA₄ on PMN migration in theapical-to-basolateral direction was present by 45 min after addition ofPMN (0.06±0.04 vs 1.51±0.12×10⁴ PMN CE/ml for vehicle control and PMNexposed to LXA₄, respectively, p<0.05), and was maintained throughoutthe 135 min experimental period (two-factor ANOVA, p<0.01).

[0089] To assess whether pre-exposure of PMN to LXA₄ was reversible, PMNwere incubated with 10 nM LXA₄ for 15 minutes, washed twice in Ca²⁺- andMg²⁺-free HBSS, and assessed for their ability to migrate acrossmonolayers of T84 epithelial cells in the apical-to-basolateraldirection. Here, PMN exposed to LXA₄ did not differ from control intheir ability to migrate across T84 epithelial monolayers (14.1±0.4 vs14.5±0.3×10⁴ PMN CE/ml for control and LXA₄ exposure followed bywashout, respectively, n=6 each, n.s.). In the presence of LXA₄, a totalof 18.6±1.0×10⁴ PMN CE/ml migrated (n=6, p<0.025 compared to control andwashout control), suggesting that LXA₄-induced enhancement of PMNmigration in the apical-to-basolateral direction requires the presenceof LXA₄.

[0090] A fmal determination was whether PMN-conditioned LXA₄ andepithelial-conditioned LXA₄ maintained its ability to enhance PMNmigration in the apical-to-basolateral direction. Samples of LXA₄ (10nM) were incubated with either PMN or T84 epithelial cells for 15 or 45minutes. Supernatants were harvested and subsequently exposed to PMN for15 min and added to the apical surface of T84 monolayers undertransmigratory conditions (1 μM fMLP transepithelial gradient) for 2 hrsat 37° C. Compared to PMN pre-exposed to HBSS (11.3±2.2×10⁴ total PMNCE/ml), PMN pre-exposed to PMN-conditioned LXA₄ (15 min) resulted in atotal PMN migration 18.0±2.1×10⁴ PMN CE/ml (n=3, p<0.05 compared tocontrol). PMN pre-exposed to epithelial-conditioned LXA₄ (15 min)resulted in a total PMN migration 16.8±3.3×10⁴ PMN CE/ml (n=3, p<0.05compared to control). Supernatants from PMN-conditioned LXA₄ (45 min)were not effective in enhancing PMN migration in theapical-to-basolateral direction (12.36±3.1×10⁴ total PMN CE/ml comparedto HBSS control of 14.3±3.2×10⁴ total PMN CE/ml, n=3, p=n.s.). Theseresults suggest that enhancement of PMN migration in theapical-to-basolateral direction involves a step which is subsequent toPMN pre-incubation with LXA₄.

PMN Pre-exposure to LXA₄ Decreases fMLP-induced PMN Migration In theBasolateral-to-aqpical Direction

[0091] Quantitative as well as qualitative differences can exist in PMNtransepithelial migration depending on the direction of migration. Toinvestigate the effect of LXA₄ on the polarity of migration, invertedmonolayers (which permit basolateral-to-apically directed migration)were prepared.

[0092] PMN (1×10⁷/ml) were pre-incubated with 10 nM LXA₄ for 15 min. at37° C. and layered on the basolateral surface of washed T84 epithelialmonolayers (i.e. inverted monolayers) at a density of 4×10⁵/monolayer.PMN were driven to transmigrate apically under the influence of a 1 μMgradient of fMLP.

[0093] Results were obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) from lower reservoirs and washed monolayers after120 min, relative to a known standard number of PMN. Since tightjunctions are the rate limiting barrier to passive paracellularpermeation, transmigration is defined as movement of PMN across thetight junction. Since monolayer-associated PMN were largely below thetight junction, total transmigration in the basolateral-to-apicaldirection equates with PMN in the opposite reservoir only. Data werepooled from 9 individual monolayers in each condition and results wereexpressed as the mean and SEM.

[0094] Pre-exposure of PMN to LXA₄ (10 nM) for 15 minutes markedlydecreased PMN migration in the basolateral-to-apical direction. Unlikethe results found in the apical-to-basolateral direction, migration ofPMN's in this physiologically relevant direction was significantlydecreased compared to vehicle controls (28.02±3.08 vs 18.77±1.48×10⁴PMN/ml for control and PMN pre-exposed to 10 nM LXA₄ for 15 min,respectively, p<0.01). Migration in the basolateral-to-apical directionresulted in no significant difference in the number ofmonolayer-associated PMN's following pre-exposure to LXA₄ (2.01±0.20 vs2.00±0.31 for control and PMN exposed to LXA₄, respectively, p=n.s.).This polarized action of LXA₄ was confirmed by performing parallelapical-to-basolateral and basolateral-to-apical migration experimentsusing T84 cells from the same plating and same passage and using PMNfrom the same donors on three separate occasions.

[0095] A time course of LXA₄ pre-exposure to PMN was next performed forbasolateral-to-apical directed migration. PMN (1×10^(7/)ml) werepre-incubated with various indicated concentrations of LXA₄ for 15 min.at 37° C. and layered on the basolateral surface of washed T84epithelial monolayers at a density of 4×10⁵/monolayer. PMN were drivento transmigrate basolaterally under the influence of a 1 μM gradient offMLP.

[0096] Results were obtained by harvesting the PMN specific enzymemyeloperoxidase (MPO) from lower reservoirs and washed monolayers after120 min, relative to a known standard number of PMN. Data were pooledfrom 9-12 individual monolayers in each condition and results areexpressed as the mean and SEM.

[0097] Similar to the apical-to-basolateral direction, decreasedtransepithelial migration was present at 15 minutes of PMN pre-exposureto 10 nM LXA₄ (11.07±1.83 for control vs. 6.29±1.21×10⁴ PMN/ml, p<0.01).No differences in the number of monolayer-associated PMN were present atany period of LXA₄ exposure. Dose-response experiments (all 15 min.pre-exposure) revealed that pre-exposure of PMN to 10 and IrM LXA₄resulted in a significantly reduced number of transmigrating PMN in thebasolateral-to-apical direction (11.07±1.83×10⁴ PMN/ml for controlsamples vs 6.29±1.21 and 6.99±1.33×10⁴ PMN/ml following pre-exposure to10 and 1 nM LXA₄, respectively, both p<0.025). Again, this diminishedtransmigratory response in the basolateral-to-apical direction wasassociated with reservoir-associated PMN only, with no apparent effecton the number of monolayer-associated PMN.

[0098] It was also determined whether PMN-conditioned LXA₄ orepithelial-conditioned LXA₄ were effective in decreasing PMN migrationin the basolateral-to-apical direction. Similar to the results in theapical-to-basolateral direction (see above), PMN pre-exposed to eitherPMN-conditioned LXA₄ (8.07±1.63 vs. buffer control 13.18±1.91×10⁴PMN/ml, n=4, p<0.025 ) or epithelial-conditioned LXA₄ (9.01±1.76compared to buffer control of 14.23±2.06×10⁴ PMN/ml, n=4, p<0.04)maintained activity which decreased PMN transepithelial migration in thebasolateral-to-apical direction.

Pre-exposure of PMN to Structurally Related Lipoxins

[0099] To investigate the specificity of LXA₄ causing decreasedmigration in the physiological direction, the effects of PMN exposure toLXB₄ and 11-trans-LXA₄ were also examined. PMN (1×10⁷/ml) werepre-incubated with 10 nM LXA₄, LXB₄ or 11-trans-LXA₄ for 15 min. at 37°C. and layered on the basolateral surface of washed T84 epithelialmonolayers (i.e. inverted monolayers) at a density of 4×10⁵/monolayer.PMN were driven to transmigrate apically under the influence of a 1 μMgradient of fMLP. Results were obtained by harvesting the PMN specificenzyme myeloperoxidase (MPO) after 120 min, relative to a known standardnumber of PMN. Total MPO activity (including reservoir- andmonolayer-associated MPO activity) was expressed as the percent PMNmigration inhibition. Data were pooled from 7 individual monolayers ineach condition and results were expressed as the mean and SEM.Pre-exposure of PMN's to 10 nM LXB₄, 11-trans-LXA₄ produced a 7±4%(p=n.s. compared to vehicle control) and 16±6% (p<0.05) inhibition ofPMN migration, respectively, while LXA₄ inhibited migration by 28±4%(p<0.01). These observations suggest structural specificity for LXA₄.

Effect of Inhibitors on LXA₄-Elicited Enhancement of PMN TransepithelialMigration In the Apical-to-basolateral Direction

[0100] To determine whether LXA₄-induced modulation of PMN migrationcould be pharmocologically altered, a series of experiments were done inwhich PMN were exposed to specific inhibitors, washed free of inhibitorand subsequently assayed for the LXA₄ effect on PMN transepithelialmigration in the apical-to-basolateral direction.

[0101] Pre-exposure of PMN to indomethacin (50 μM, 15 min, 37° C.), acyclooxygenase inhibitor, (Smolen J. E., and G. Weissman (1980).Biochem. Pharmacol. 29: 533-538), did not effect baseline PMN migrationin the presence of a transepithelial gradient of fMLP (109±13% vehiclecontrol, n=6, p=n.s. compared to untreated control). Likewise,pre-exposure of PMN to indomethacin followed by exposure to LXA₄ (10 nM)did not alter the LXA₄-elicted increase in fMLP-driven PMN migration inthe apical-to-basolateral direction (61±11% increase vs 54±7% increaseover control for LXA₄ treated PMN with and without indomethacin,respectively, p=n.s.). Likewise, PMN pre-treatment with the compoundMK886 (10 ng/ml), a specific inhibitor of leukotriene generation,(Gillard J., et al. (1989). Can. J. Physiol. Pharmacol. 67: 456-464),did not alter baseline fMLP-driven PMN migration and did not effect theLXA₄-elicited increase in PMN transepithelial migration.

[0102] Staurosporine, a potent inhibitor of protein kinase C (PKC),(Sako T., et al. (1988). Cancer Res. 48: 4646-4650), was assessed forits ability to inhibit the LXA₄ effect. Interestingly, staurosporinealone (10 nM final concentration) was found to inhibit PMNtransepithelial migration (93±5% inhibiton vs.vehicle control, n=3,p<0.001). These data were also confirmed using the PKC inhibitor H7(Nakadate, T., et al. (1989). Mol. Pharmacol. 36: 917-924.) (100 μMfinal concentration, 91±4% inhibition compared to vehicle control, n=3,p<0.001). Likewise, the LXA₄-elicited (10 nM) increment in migration wassensitve to staurosporine (89±7% inhibition compared to vehicle control,n=3, p<0.001). Pre-exposure of PMN to pertussis toxin, Nigam, S., et al.(1990). J. Cell Physiol 143:512-523), (2 μg/ml) also inhibited baselinefMLP driven migration (91±8% inhibition compared to vehicle control,n=6, p<0.001). The LXA₄-elicited (10 nM) increase in PMN migration(46±3% increase compared to control, p<0.01) was also sensitive to PMNpre-exposure to pertussis toxin (87±4% inhibition of control, n=6,p<0.001).

[0103] Next assessed was the possibility of differential sensitivity tostaurosporine for baseline and LXA₄-stimulated increases in fMLP-drivenPMN migration. Staurosporine inhibited baseline PMN transepithelialmigration in a dose-dependent manner (94±4%, 96±9%, 67±11%, 54±8%,35±11% and 11±6% inhibition compared to vehicle for concentrations of100, 10, 1, 0.1, 0.01 and 0.001 nM staurosporine, respectively, p<0.01by ANOVA). From this dose response, a concentration was selected whichwas approximately half-maximal in inhibiting PMN migration (0.1 nM, seeabove). PMN were then pre-exposed to staurosporine (0.1 nM, 15 min, 37°C.), washed free of inhibitor, and subsequently assessed for the LXA₄effect on PMN transepithelial migration. Here, the LXA₄-elicitedincrease in transepithelial migration of PMN was observed to besensitive to PKC inhibition, since the relative inhibition bystaurosporine was equivalent with and without LXA₄ (54±6% and 49±7%decrease in total PMN migration for staurosporine treated PMN in thepresence and absence of LXA₄, respectively, p=n.s.; both decreasedcompared staurosporine untreated controls, n=6, p<0.025).

[0104] These results indicate that LXA₄-elicited increases in PMNmigration in the apical-to-basolateral direction are not sensitive toinhibition of the cyclooxygenase pathway or the specific inhibitionleukotriene generation, but is sensitive to inhibitors of PKC.

[0105] Discussion

[0106] During inflammatory processes, PMN are recruited from the bloodby signals derived at inflammatory sites. At sites of acuteinflammation, PMN function may be regulated by a variety of inflammatorysignals, including both protein- and lipid-derived signals. PMN functionat organ-specific sites, including the intestine, are thought tocontribute to epithelial dysfunction during disease. Here it is reportedfor the first time that the arachidonic acid-derived eicosanoid, LXA₄,modulates PMN migration across a model human intestinal epithelium. Inaddition, it is here reported that LXA₄ exerts an effect on migration ina polarized fashion.

[0107] LXA₄ enhances PMN transepithelial migration in theapical-to-basolateral direction. For technical reasons, previous studiesof PMN transepithelial migration have focused on “non-physiologically”oriented monolayers, in which leukocyte migration is in theapical-to-basolateral direction, (Nash S., et al. (1987). J. Clin.Invest. 80: 1104-1113), (Migliorisi G. E., et al. (1988). J. LeukocyteBiol. 44: 485-492), (Parkos C. A., et al. (1991). J. Clin. Invest. 88:1605-1612), (Evans C. W., et al. (1983) Br. J. Exp. Pathol. 64:644-654).PMN pre-exposed to LXA₄ and driven to transmigrate across epithelialmonolayers oriented non-physiologically resulted in enhanced PMNmigration.

[0108] The action of LXA₄ was found to be specific for PMN, and notepithelial cells, since enhanced PMN migration in this direction wasdose- and time-dependent, and no measureable effects on PMNtransepithelial migration were observed when epithelial monolayers werepre-exposed to LXA₄ under conditions which promoted enhanced PMNmigration. These results are consistent with previous studies whichreport that LXA₄, in similar concentrations used here, was capable ofactivating PMN in vitro. In this model system, LXA₄ enhanced PMNmigration in a manner independent to that of fMLP, since in allconditions PMN migration was driven toward a gradient of fMLP,suggesting that the proportion of PMN migration exceeding that of fMLPcontrols is dependent on a LXA₄-mediated event. Moreover, these resultssuggest that the action of LXA₄ may be synergistic with fMLP, sinceLXA₄, by itself, does not promote PMN migration in the absence of fMLP.

[0109] In addition, it was discovered that pre-exposure of PMN to LXA₄modulates migration of PMN in a polarized manner. That is, oppositeeffects were observed depending on the direction of PMN migration. Theobserved effect of LXA₄ inhibition of PMN migration in thephysiologically oriented (basolateral-to-apical) direction was dependenton concentration—as well as the duration of pre-exposure. These effectswere found to be selective for LXA₄, since no effect was observed withthe positional isomer LXB₄.

[0110] As described for leukocyte movement across endothelia, (ButcherE. C. (1991) Cell 67: 1033-1036), PMN migration across epithelialmonolayers is likely a multi-step process requiring engagement anddisengagement of several receptor-ligand complexes between PMN andepithelial cell. The specific events involved in PMN transepithelialmigration are poorly understood at the present time, but in partrequires the PMN B2 integrin CD11b/CD18 and is independent of ICAM-1.(Parkos C. A., et al. (1991). J. Clin. Invest. 88:1605-1612). Inaddition, PMN transepithelial migration can be regulated by exposure ofT84 epithelial monolayers to the lymphokine interferon-gamma (IFN-γ). Inlight of the polarized nature of this epithelium, (Madara, J. L., and K.Dharnsathaphom (1985). J. Cell Biol. 101:2124-2133), (Madara J. L., etal. (1987). Gastroenterology 92:1133-1145), (Dharmsathaphom K., and J.L. Madara (1990). Meth. Enzymol. 192: 354-389), it would not besurprising that the sequence by which PMN encounters epithelial ligandsdirectly regulates PMN migration.

[0111] Moreover, PMN migration across endothelia requires a sequentialseries of activation and deactivation steps on the PMN surface, of whichlipid-derived activating factors may play an important role (reviewed inButcher E. C. (1991) Cell 67:1033-1036). Whether LXA₄ could act as alipid-derived factor for expression of a crucial ligand in theregulation of PMN migration across epithelia is not known. Evidence tosupport this hypothesis are provided by a recent study characterizinglipoxin binding sites on human PMN. (Palmblad J., et al. (1987) Biochem.Biophys. Res. Commun. 145: 168-175). With a reported K_(d) of 0.5 nM andapproximately 1800 binding sites/cell, the range of LXA₄ concentrationsused in the present study (0.01-10 nM) should provide maximal activationof subsequent signal transduction steps, most of which remain to beelucidated, but appears to involve a G-protein associated activationstep (Fiore S., et al. (1992) J. Biol Chem. 267: 16168-16176), andpossibly a signalling step through PKC as determined by inhibition usingstaurosporine.

[0112] Recent in vivo studies have shown that LXA₄ is an importantlipid-derived mediator at several distinct anatomic sites, including thelung, (Christie P. E., et al. (1992) Am. Rev. Respir. Dis. 145:1281-1284), kidney, (Katoh T., et al. (1992) Am. J. Physiol 263:F436-F442), blood vessel (Brezinski D. A., et al. (1992) Circulation.86:56-63), and hamster cheek pouch (Hedqvist P., J. et al. (1989). Acta.Physiol. Scand. 137:571-572). The data reported here suggest thatLXA₄-elicited alterations exert effects.at the level of PMN andsubsequently modulate PMN-epithelial interactions. Previous studies haveshown that production of lipoxygenase products of arachidonic acidcorrelate with intestinal inflammation. Specifically, an enhancedconversion of arachidonate to 5-, 12- and 15-hydroxy-eicosatetraenoicacid (HETE) has been shown in ulcerative colitis homogenates(Broughton-Smith, N. K., et al. (1983) Gut 24:1176-1182), as well asincreased biosynthesis of LTB₄ in Crohnls disease (Sharon, P. and W. F.Stenson (1984) Gastroenterology 86:453-460). The present resultsdemonstrate that lipoxins, and especially by their action on PMN, play arole in intestinal disease.

Example 2 Measurement of Electrical Parameters of Cultured EpithelialMonolayers: Use in Assessing Neutrophil-Epithelial InteractionsMaterials and Methods

[0113] General information regarding T84 cells: T84 cells were negativefor mycoplasma as tested commercially using a nucleic acid probe(Organon TeKnika Corp., Rockville, Md.). T84 cells were first obtainedfrom Dharmsathaphorn in 1984 (Dharmsathaphorn, K. Am. J. Physiol. 246(Gastrointest. Liver Physiol. 9):G204-G208; 1984). T84 cells wereoriginally isolated from a lung metastasis of a patient with coloniccarcinoma and were established as a transplantable line in BALB/c nudemice (Dharmsathaphorn, K. Am J. PhysioL 246 (Gastrointest. LiverPhysiol. 9):G204-G208; 1984). However, T84 cells are now available fromthe ATCC (American Type Culture Collection, Rockville Md., cat #CCL248). ATCC derived T84 cells passages 60-100 have been compared with theoriginal distributed parent line (passages 1640) by assayingelectrogenic Cl⁻ secretory responses to cAMP (8-bromo-cAMP,theophylline, or forskolin) and Ca⁺² (ionomycin, A23187, carbachol)agonists, cell migration patterns, neutrophil transmigration, andcytoskeletal responses to bacterial (C. difficile toxin A) and fungal(cytochalasin D) derived toxins. The cells from these two sources arehighly comparable. Some differences between T84 cells from these twosources do exist in conditions for loading cells with reagents whichcross the plasma membrane passively (such as loading phalloidin intoliving cells (Shapiro, M. et al; J. Clin. Invest. 87:1905-1909; 1991)).However, even within cells from ATCC, optimal loading conditions canvary substantially over 20-25 passages and thus loading conditions withcells from any source must be empirically defined in each laboratory.

[0114] Growth and Maintenance of T84 cells: As previously detailed(Dharmsathaphorn, K.; Madara, J. L. Meth. Enzymol. 192:354-389; 1990),T84 cells are grown as monolayers in a 1:1 mixture of Dulbecco-Vogtmodified Eagle's (DME) medium and Ham's F-12 medium supplemented with 15mM Na+-HEPES buffer, pH 7.5, 1.2 g/l NaHCO₃, 40 mg/liter penicillin, 8mg/liter ampicillin, 90 mg/liter streptomycin, and 5% newborn calfserum. Growth requirements are not strict as cells will grow in avariety of media (Dharmsathaphom and Madara, personal observations,(Dharmsathaphorn, K.; Madara, J. L. Meth. Enwmol. 192:354-389; 1990).However, some care must be taken in selecting media, since mediaselected strictly for the ability to increase growth rate can result inthe loss of the polarized phenotype (Madara, personal observations).Cells are split near confluency by incubating in 0.1% trypsin and 0.9 mMEDTA in Ca+2 and Mg+2 free phosphate buffered saline for 15-20 min. T84cells grow best when split 1:2 and plating at lower densities maygreatly retard growth. After splitting, cells are generally nearconfluency once again in 5-8 days; thus they are relatively slow growingcells. T84 cells aggregate in suspension and attempts to produce uniformdispersal of cells will result in substantial loss of viability andresulting low effective plating density.

[0115] Preparation of monolayers: Normal or inverted monolayers can beconstructed for a physiological microassay using the commerciallyavailable insert system (Costar inserts, 0.33 cm², 5μm polycarbonatefilters). The larger pore size is crucial for allowing neutrophils topenetrate the filter. Collagen 1 must coat the filters to allowattachment of T84 cells. Original descriptions of the collagen coatinvolved procedures in which the collagen was chemically crosslinkedDharmsathaphom, K. et al; Meth. Enzymol. 192:354-389; 1990.Dharmsathaphorn, K. et al; Am. J. Physiol. 246 (Gastrointest. LiverPhysiol. 9): G204-G208; 1984. -However, neutrophil movement acrosscrosslinked collagen gels has been found to be extremely limited.

[0116] To circumvent this problem, Cereijido and Sabatini's method(Cereijido, M.; Sabatini, D. D. 1978 J. Cell Biol. 77:853-876;) ofpreparing viscous collagen from rat tail tendons is followed and thissolution is mixed 1:3 with 60% ethanol at 4° C. Very little collagen isrequired; indeed collagen ethanol ratios of 1:100 can be readily used.Fifty microliters of this mixture is then placed on each filter, takingcare of an even distribution and plates are allowed to dry in a hood (3hr-overnight). A few drops of media are then added to the wells for 1-3hr, and cells (10⁶/cm²) are then added in a total volume of 167 μl.Eight-hundred microliters of media are added to the outer wells and afew drops of additional fresh media to the inner well.

[0117] After reaching the initial steady state resistance, themonolayers should be used within 6-14 days for two reasons: first,physiological responses such as C1-secretion will diminish with time andsecond, without underlying crosslinked collagen, cell processes caneventually move through the 5μM pores and ultimately result in a neardouble monolayer (monolayer on each side of the filter). Monolayers needonly have one feeding, but this should take place at least 24 hrs priorto experimental use.

[0118] Inverted monolayers can also be grown using this technique. Forthis 0.8 mm thick lexan rings having the same dimension of the base ofCostar inserts are machined, deburred, cleaned by boiling with a traceof detergent and subsequently exhaustively washed, and attached to theunderside of the insert using General Electric RTV Silicone glue (thisunderside ring is necessary for a peripheral electrical seal). Afterdrying overnight, the inserts are sterilized by submersion in 70%ethanol (4-hr overnight), inverted onto a sterile petri dish in a hood,and allowed to dry. Collagen and cells are added to the filter(underside now facing up) exactly as with unmodified inserts and cellsare allowed to attach for 4 hours before righting the inserts into the24 well holding plates. Subsequent treatment of the monolayers isidentical to that described above.

[0119] Isolation of neutrophils: Neutrophils are isolated from wholeblood using a gelatin-sedimentation technique. Briefly, whole blood,anticoagulated with citrate/dextrose, is centrifuged at 300× g for 20minutes (20° C.). The plasma and buffy coat are carefully removed usinga curved, siliconized glass pipette which is attached to a vacuum trap.Care must be taken not to aspirate the interface between the buffy coatand RBC since this is where PMN reside. To eliminate contaminating RBC,a 2% solution of gelatin (100 bloom, Fisher) made up in either saline orHBSS (35-40° C.) is added to the RBCIPMN mixture at a ratio of 35 ml ofgelatin per 15-20 ml of cells.

[0120] The gelatin/cell mixture is then incubated at 37° C. for 30minutes to settle-out contaminating RBC. The pink supernatant is thencentrifuged at 400× g for 10 minutes (20° C.) to yield a red pellet ofPMN and some RBC's. Residual RBC's are then lysed by gentle resuspensionof the pellet with isotonic ammonium chloride (t=4° C.), followedimmediately by centrifugation at 300× g, 10 minutes, 4° C. After washingtwice in HBSS (HBSS without Ca¹⁺² or Mg⁺²), the cells can be counted andresuspended at 5×10⁷ PMN/ml and are ready for use. The above methodusually yields 1−2×10⁸ PMN from 100 ml of whole human blood at a purityof approximately 90%.

[0121] Physiological assays: All solutions and materials are maintainedat 37° C. A convenient way to do this is to perform the experiments in a37° C. room. T84 cells do well in this environment in bicarbonate freebuffers for at least four hours. Hanks Balanced Salt Solution (HBSS,Sigma, without bicarbonate or phenol red) to which 10 mM HEPES buffer isadded, pH 7.4 is used for these assays. Inserts with attached monolayersare lifted from wells, drained of media by inverting, and gently rinsedby dipping in a 200 ml container of HBSS. Inserts are then placed in newwells with 800 μl fMLP in the lower compartment and subsequently 100 μlHBSS is added to the inner well (an additional 100 μl containingneutrophils in HBSS is added to the inner well to initiate theexperiment). The above treatment has little effect on resistance butwashing or other trauma does consistently result in a transienttransepithelial current (4-15 uA/cm2) which returns to baseline (0-3μA/cm²) within 4 minutes.

[0122] To measure currents, transepithelial potentials, and resistance,the following system was utilized: a commercial voltage clamp (Iowa DualVoltage Clamps, Bioengineering, University of Iowa), interfaced with anequilibrated pair of calomel electrodes submerged in saturated KCl andwith a pair of Ag-AgCl electrodes submerged in HBSS. Agar bridges arethen made: HBSS containing 6% agar is heated in a water bath until theagar is in solution and the solution is perfectly clear. Using a syringefor suction, this hot solution is then pulled through 1 mm borepolyethylene tubing (12 cm lengths), the agar is allowed to gel, and theends are trimmed to a 45 tapper with a razor blade. Agar bridges arethen used to interface the electrodes with the solutions on either sideof the monolayers (one calomel and one Ag-AgCl electrode in each well).

[0123] The agar bridge pair to the inner well is properly positionedwhen the surface tension of the fluid above the monolayer is broken. Thepair of bridges in the outer well is positioned by inserting the agarbridges to the bottom of the well (through one of the openings on theside of the insert) and then withdrawing it 1 mm. In practice, ahand-held polycarbonate strip, which fixes all distances and positions,had been made. Such a bridge-holding device makes use of the fact thatall distances are fixed (from top of well to monolayer, from side ofwell to insert center, etc.). In high resistance monolayers, such asT84, positional effects are minimal. This is likely due to therelatively high resistance of the monolayer which promotes relativelyuniform current densities at the monolayer surface. The taper in thetips of the agar bridges is also advantageous in preventing entrapmentof air bubbles when gently positioning the inner bridges and inpreventing abutment of the outer bridges with the plate bottom whichleads to spuriously high resistance readings.

[0124] For measurements, bridges are positioned as described, and thespontaneous transepithelial electrical potential and the instantaneouspotential generated by passing 25 μA of current are measured. Similarmeasurements are taken after scraping the filter with a pipette andthese later measurements are used to correct for system resistance.Using these values and Ohm's Law, tissue resistance and transepithelialcurrent are then calculated. System resistance is less than 5% of thetotal resistance value. In a single monolayer in which the bridges weresimply held by hand and repositioned 20 times (making sure that bothelectrode pairs were varying in position) resistance and spontaneoustransepithelial electrical potential varied by less than 10%. In settingup inverted monolayers for study one must be cautious that small airbubbles are not trapped under the monolayer by the added ring. Thisproblem can be circumvented by lowering the washed insert into the HBSSof the outer well slowly and at an angle.

[0125] In practice the above approach allows one to serially recordplates of monolayers and accurate readings from 36 monolayers can beobtained in less than 10 minutes.

[0126] Myeloperoxidase assay of neutrophil transepithelial migration:PMN contents of monolayers and lower chambers can be quantitated byassaying the PMN-specific azurophil granule marker, myeloperoxidase(MPO). Monolayers are cooled to 4° C. and washed with HBSS using apipette to remove non-adherent PMN. Washed monolayers are then placed innew 24-well tissue plates and overlaid with 1.0 ml 0.5% Triton X-100 inHBSS in order to solubilize PMN-associated MPO. After 10 minutes ofvigorous shaking, monolayers can then be discarded and supernatant savedfor assay. To the original lower chambers, MPO can be solubilized bysimply adding 50 μl of 10% Triton X-100 and mixing.

[0127] To assay for solubilized MPO, the pH of solubilized chambers andmonolayers must be adjusted to 4.2 using 100 μl of 1.0 M citrate pH 4.2.Aliquots of each pH adjusted sample can then be transferred to a 96-wellmicrotiter plate for substrate addition. After adding 100 ul of asolution of 2 mM 2,2′-azino-di-(3-ethyl) dithiazoline sulfonic acid(ABTS), 0.06% H₂O₂ in 100 mM citrate buffer pH 4.2 to each well, colordevelopment can be quantitated on a microtiter plate reader at 405 nm.The reaction can be terminated by the addition of sodium dodecyl sulfateto a final concentration of 0.5%. For standards, serial dilutions of thesame PMN used in the experiment can be made in 1.0 ml of HBSS. MPO issolubilized identically as for the lower chambers. When performed inthis manner, the assay is linear in the range of 0.3−50×10⁴ cells/ml.

[0128] Results

[0129] Steady state resistance was reached within 7 days of plating andvalues greater than 1,000 ohm cm² were achieved. Both the time courseand value of resistance are comparable to that achieved on matrices ofcross-linked collagen (Omann, G. M. et al.; Physiol. Rev. 67:285-322;1991). Passage-related variation in time to reach physiologic confluence(4-7 days) and in resistance value in the steady state (500-1900 ohmcm²) does occur. Resistance is measured by the simple bridge methoddescribed. The time course and steady state resistance value achieved iscomparable to that previously published for monolayers on thickcross-linked collagen I matrices measured by formal Ussing chamber means(Madara, J. L; Dharmsathaphorn, K. J. Cell Biol. 101:2124-2133; 1987).The collagen matrix does not inhibit epithelial transmigration byneutrophils and thus is suitable for such assays. The approach tomeasurement of resistance allows one to obtain sequential values fromnumerous monolayers over a short duration of time.

[0130] Neutrophil migration across T84 monolayers was accompanied by asignificant decrease in transepithelial resistance when migration was inthe apical to basolateral direction. The size of the resistance decreasewas paralleled by the density of applied neutrophils. Neutrophildensities in cell number/cm² are indicated. Transmigration (apical tobasolateral) was stimulated by a 10⁻⁷ M transepithelial gradient offMLP. The decrease in resistance due to penetration of intracellulartight junctions by neutrophils was large at the highest neutrophildensity and is saturated within 60 minutes. Furthermore, as indicated bythe myeloperoxidase assay, the log of resistance correlates well withthe number of neutrophils migrating into the membrane (cells havingcrossed the tight junction but remaining above the filter) and with thetotal number of transmigrated cells. The log of the final resistancevalue (5×10⁶ neutrophils/cm² in 10⁻⁷ M fMLP gradient for 110 min)correlates with the number of neutrophils transmigrated. Variations inelectrical responses are due to variations in efficacy oftransmigration.

[0131] The decrease in resistance was only modest in the absence of agradient and under these conditions the number of transmigratingneutrophils was small. Antibody to the common beta chain of neutrophilβ₂ integrins (CD18) blocks transmigration and the fall in resistancewhile a control antibody (J5) recognizing CD10 has no inhibitory effect.The fall in resistance seen in controls (no fMLP, no PMN) and in thepresence of anti-CD18 antibody represents, in large part, the 200-300ohm fall in resistance occurs when monolayers are transferred from mediato HBSS.

[0132] FMLP was effective in stimulating transmigration of neutrophilsat both 10⁻⁶ and 10⁻⁷ M as measured by either resistance ormyeloperoxidase assay. TABLE 1 NEUTROPHILS MIGRATE ACROSS INVERTED,SPARSELY COLLAGEN- COATED INSERTS AND SUBSEQUENTLY ACROSS T84 MONOLAYERS(i.e. BASOLATERAL TO APICAL) Neutrophil Cell Equivalents × 10⁴* InOpposite In Monolayers Reservoir Total No FMLP⁺ 0.23 0.18 0.41 No PMN 00 0 FMLP + PMN 5.47 85.30^(#) 90.77^(#) FMLP + PMN + αCD18 1.22 5.767.00

[0133] Additionally, the blocking antibody to CD18 inhibited neutrophiltransmigration nearly completely as assessed by each assay, whilesubstantial transmigration proceeded in the presence of control antibody(anti-CD10). Transmigration which is inhibitable by antibody to CD18also occurred in the basolateral to apical direction (Table 1).Neutrophils applied to the basolateral surface of monolayers haveeffects on resistance independent of transmigration and thus themyeloperoxidase assay is most useful in this direction as a quantitativemeasure of transmigration.

[0134] As previously reported in Nash, S. et al; J. Clin. Invest.87:1474-1477; 1991, a neutrophil derived secretagogue (NDS) activity,detected as a modest short circuit current, occurs as a consequence ofneutrophil-T84 cell interactions. Such currents are readily detected inthe microassay described, are important since they represent activeelectrogenic transepithelial transport events, and are not readilydetected using “chop-stick” type detection systems.

[0135] Discussion

[0136] Detailed here are useful approaches in utilizing T84 cells forstudies of epithelial monolayer function in general andepithelial-neutrophil interactions in particular. Electrical dataobtained from Ussing chambers designed for cultured monolayers(Dharrnsathaphom, K.; Madara, J. L., Meth. Enzymol. 192:354-389; 1990.Nash, S. et al; J. Clin. Invest. 87:1474-1477; 1991. Dharmsathaphom, K.et al; Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9):G204-G208;1984) are highly accurate. However, in using such systems one is limitedby the number of such chambers available, each of which is occupied byone monolayer for the length of the experiment. Additionally, suchsystems have traditionally been designed to accommodate monolayers withlarge surface areas (such as 2 cm²) since larger surface areas providefor more accurate transepithelial flux measurements. However, fluxmeasurements are often only required to address specific issues and thebulk of many experiments can often be completed with simple electricalassays of resistance and short circuit current.

[0137] Electrical data with reproducibility rivaling that of moreformalized but tedious systems can be obtained, in high resistanceepithelial monolayers such as T84, by simply interfacing electricalmeasuring systems usually utilized in more formal Ussing chambersystems, with a commercially available insert system. It would not besurprising if the electrical data derived from this system were of lessquality in low resistance monolayers, since imposed current densities atthe monolayer surface would likely be less uniform and thereforepositional effects could be more problematic. Moreover, it is not likelythat the insert system will be suitable for non-electrical assays ofpermeability such as flux. Major limitations in this regard are the lackof stirring and the overly small compartment (apical) from which toobtain samples.

[0138] Also described is a simple procedure for manufacturing invertedmonolayers so that neutrophils or other cells can settle by gravity onthe basolateral side of the monolayer. In this system, attention tocomposition of the matrix on which one plates cells determine whethercells and macromolecules added to the basolateral surface are able togain access to the basolateral membrane of the epithelial cell. Lastly,a simple enzymatic assay which allows quantitation of numbers ofneutrophils which have migrated either into or across epithelialmonolayers is described.

[0139] The data presented, which show examples of the application ofthese approaches to types of studies previously assessed in formalizedUssing chamber experiments, indicate that such methods are useful fordetecting the physiologic effects of neutrophil-epithelial interactionson epithelial barrier function and activated transepithelialelectrogenic transport. The approach outlined can be used to show thatintestinal epithelial barrier function is diminished and Cl⁻ secretionis activated by such neutrophil-epithelial interactions. Both of thesefunctional disorders of intestinal epithelia are known to occur in vivoin humans affected by intestinal diseases characterized by neutrophilinfiltration of the epithelium. Thus, such in vitro cell culture modelsof the interactions between these two cell types provide an opportunityfor mechanistic studies of the symptomatology occurring in theseimportant human diseases,

Example 3 Synthesis of Lipoxin Analog Compounds

[0140]

Preparation of the Methyl Ester Precursor of Compound 1

[0141] To a solution of 3-methyl-3-trimethylsiloxy-1-bromo-1-octene (130mg. 0.44 mmol) in benzene (1.5 mL) was added n-propylamine (0.05 mL,0.61 mmol) and Pd(PPh₃)₄ (20 mg. 0.02 mmol) and the solution wasprotected from light. It was then degassed by the freeze-thaw method andstirred at rt for 45 min. (7E, 9E, 5S, 6R) Methyl5,6-di(tert-butyldimethylsiloxy)-dodeca-7,9-diene- 1I-ynoate (183 mg.0.44 mmol) (compound 12) and copper iodide (14 mg. 0.07 mmol) were addedand the solution was one more time degassed by the freeze-thaw method.The mixture was stirred for 3 h at rt and quenched with saturatedaqueous solution of NH₄Cl and extracted with ether. It was then washedwith brine and dried over MgSO₄ and the solvent was evaporated. Flashcolumn chromatography (silica, 3% ether hexanes) afforded pure compoundas a colorless liquid (171 mg. 57% yield).

[0142] To a solution of the compound (171 mg. 0.25 mmol) in THF (0.5 mL)was added n-BuN₄F(0.9 mL. 0.90 mmol) and the mixture was stirred at rt.The reaction was completed in 2 h at which time it was poured into waterand extracted with ether. The ether extracts were washed with brine,dried over Na₂SO₄ and the solvent was evaporated. Flash columnchromatography (silica 4% MeOH/CH₂Cl₂) afforded the methyl ester (24mg.) together with some of the corresponding lactone. HPLC retentiontime: 9:39 min (microsorb reverse phase, 4.6mm×25 cm, C-18 column,MeOH/H₂O 70:30 flow rate 1 ml/ min, WV detector at 300 nm). WV in MeOH:γ _(max)283, 294, 311 nm. ¹HNMR (500 MHz CDCl₃) δ6.53 (dd. 15.2 10.9 Hz,1 H), 6.32 (dd, J=15.1, 11.0 Hz, 1 H), 6.17 (d, J=15.9 Hz, 1 H) 5.83(dd. J=17.5,2.1 Hz, 1 H), 5.80 (dd. J=15.2, 6.7 Hz, 1 H), 5.72 (dd.J=17.0, 2.1 Hz, 1 H), 4.14 (m, 1 H), 3.68-3.64 (m, 4H), 2.35-2.31 (m, 2H), 1.51-1.48 (m, 1 H), 1.43-1.42 (m, 2 H), 1.30-1.23 (m, 15 H) 0.85 (t,3 H). ¹³ C NMR (126 MHz, CDCl₃) δ150.01, 140.18, 132.95, 132.26, 112.43,107.50, 75.23, 73.76, 42.49, 33.67, 32.17, 31.36, 27.96, 23.56, 22.58,21.03, 14.03.

Preparation of the MethyI Ester Precursor of Compound 2

[0143] A solution of the methyl ester precursor of compound 1 (3 mg. inCH₂Cl₂ (1 ml) was mixed with Lindlar's catalyst (1 mg.) and placed undera hydrogen atmosphere. The mixture was stirred at rt in the darkfollowed by HPLC until about 80% conversion (1 h). Filtration overcelite evaporation of the solvent and separation by HPLC gave a puremethyl ester. HPLC retention time: 10:02 min (microsorb reverse phase.10 mm×25cm C-18 column, MeOH/H₂O 70:30 flow rate 4 ml/min. UV detectorat 300 nm). UV in MeOH: η_(max) 287, 301, 315 nm.

Preparation of the Methyl Ester Precursor of Compound 3

[0144] This compound was prepared similarly to the preparation of themethyl ester precursor of compound 1 (from3-cyclohexyl-3-trimethylsiloxy-1-bromo-1-octene). Desilylation of thiscompound was also performed in a similar manner to afford the methylester. HPLC retention time 8:02 min (microsorb reverse phase, 4.6 mm×25cm. C-18 column, MeOH/H₂O 70:30, flow rate 1 mmin, UV detector at 300nm). UV in MeOH: λ_(max) 282, 293, 311 nm. ¹H NMR (360 MHz, CDCl₃) δ6.56(dd, 15.4, 10.9 Hz, 1 H), 6.33 (dd, J=15.2, 10.9 Hz, 1 H), 6.13 (dd,J=15.8, 6.5 Hz, 1 H), 5.81 (dd, J=15.2, 6.4 Hz, 1 H), 5.80 (d, J=15.6Hz, 1 H), 5.73 (dd, J=15.4, 2.1 Hz, 1 H), 4.15 (br, 1 H), 3.93-3.90 (m,1 H), 3.67 (br, 1 H), 3.65 (s, 3 H), 2.34 (t, 2 H), 1.82-1.65 (m, 10 H),1.46-1.38 (m, 3 H), 1.26-1.01 (m, 5 H).

Preparation of the Methyl Ester Precursor of Compound 4

[0145] Selective hydrogenation of the methyl ester precursor of compound3, followed by HPLC purification gave the methyl ester precursor ofcompound 4. HPLC retention time: 9.72 min (microsorb reverse phase, 10mm×25 cm C-18 column, MeOH/H₂O 70:30 flow rate 4 ml./min. UV detector at300 nm), UV in MeOH: λ_(max) 288, 301, 315 nm. ¹H NMR (250 MHz, C₆D₆) δ6.66-6.89 (m, 2 H), 5.95-6.24 (m, 4 H), 5.55-5.66 (m, 2 H), 3.82 (m, 1H), 3.73 (m, 1 H), 3.41 (m, 1 H), 3.31 (s, 3H, OCH₃), 2.08 (t, 2 H,CH₂COO), 1.00-1.81 (m, 18 H).

[0146] The methylesters can be converted to corresponding alcohols usingstandard techniques.

Synthesis of 15(R)-15-methyl-LXA₄ and 15(±)methyl-LXA₄

[0147] Approximately 1 gm acetylenic ketone a is prepared usingFriedel-Crafts acylation of bis(trimethylsilyl) acetylene with hexanoylchloride and is reduced using (−)-pinayl-9-BBN to give the (S) alcoholin CH₃N₂ as in Webber, S. E. et al. (1988) Adv. Exp. Med. Biol. 229:61;Nicolaou, K. C. et al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100; andVorbruggen, H. et al.: In: Chemistry, Biochemistry, and PharmacologicalActivity of Prostanoids (Roberts, S. M., Scheimnann, F. eds.). Oxford:Pergamon Press, to generate the methyl at C-15.

[0148] Alternatively, the keto group can be treated with CH₃MgBr (60

70° C.) as in Vorbriiggen, H. et al.: In: Chemistry, Biochemistry, andPharmacological Activity of Prostanoids (Roberts, S. M., Scheinmann, F.eds.). Oxford: Pergamnon Press to yield the 15(±)methyl of b (2-5 g) indry CH₂Cl₂ (˜20 ml) at 0° C. with sequential additions of 2,6-lutidine(5.2 ml) and tert-butyldimethylsilyl triflate (6.9 ml). This reaction ismixed for 1 h and then diluted with 100 ml ether for aqueous extractionand drying with MgSO₄.

[0149] The product c is then coupled with d

[0150] that is generated as in Nicolaou, K. C. et al. (I199 1) AngewChem. Int. Ed. Engl. 30:1100; Nicolaou, K. C. et al. (1989) J. Org.Chem. 54:5527 and Webber, S. E. et al. (1988) Adv. Exp. Med. Biol.229:61. Structure d from fragment A in Scheme I is suspended in 4.0equiv. of AgNO₃, then 7.0 equiv. of KCN, containing EtOH:THF:H₂0(1:1:1), 0-25° C. for 2 h to generate the C-methyl ester protected15-methyl-LXA₄ analog that is concentrated and saponified in THF withLiOH (2 drops, 0.1 M) at 4° C. 12-24 h to give the corresponding freeacid.

Synthesis of 16-dimethyl-LXA₄

[0151]

[0152] This compound is generated using the similar strategy by couplingd above with e vide supra, or f to generate the 15-phenyl-LXA₄ analog,or g to generate the 17-m-chlorophenoxy-LXA₄ analogs.

[0153] The appropriate C fragments in Scheme I (i.e. e, f, g, h, ) areeach prepared as reviewed in Radjichel, B. and Vorbruiggen, H. (1985)Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263 for the knowncorresponding prostaglandin analogues. In h, R=H; Cl, methoxy orhalogen.

Synthesis of 13,14-Acetylenic-LXA₄ and Halogen-containing Analogs

[0154]

[0155] Using the A₂B₂ generated fragment from Scheme II, thecorresponding C₂ fragments are prepared for coupling. Structures j and kare generated as in Nicolaou, K. C. et al. (1989) J. Org. Chem. 54:5527and methylated as in Radutchel, B. and Vorbrdggen, H. (1985) Adv.Prostaglandin Thromboxane Leukotriene Res. 14:263 are coupled to 7 toyield these LX analogues. The materials may be subject to RP-HPLC forpurification vide supra.

Synthesis of 14,15-acetylenic-LXA₄

[0156]

[0157] The designated combined A₂B₂ fragment can be prepared fromcouplings of fragments A₁ and B₁, illustrated in Route II to carry thestructure of 7 or 4 vide supra for coupling to fragment C ₂. Theprecursor for the C ₂ fragment 1 can be prepared as in Raduichel, B. andVorbruggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res.14:263 for a prostaglandin analog.

[0158] Precursor m as prepared previously (Nicolaou, K. C. (1989) J.Org. Chem. 54:5527) is added at 1.2 equiv. to 0.05 equiv. of Pd(PPh₃)₄,0.16 equiv. of Cul, n-PrNH₂, in benzene with Me₂Al-carrying 1, 2-3 h RTto yield n.

[0159] The alcohol protecting groups TBDMS=R are removed with 10 equiv.of HF-pyr, THF, 0-25° C. (4 h) followed by exposure to 3.0 equivalentsof Et₃N, MeOH, 25° C. 15 min to open acid-induced δ-lactones thatusually form between C-1-carboxy and C-5 alcohol in the lipoxins(Serhan, C. N. (1990) Meth. Enzymol.187:167 and Nicolaou, K. C. (1989)J. Org. Chem. 54:5527). After mild treatment with Lindlar cat. 5% byweight, the extracted material may be subjected to LiOH saponificationin THF to generate the free acid of the target molecule that can besubject toffurther purification by RP-HPLC gradient mobile phase as in(Serhan, C. N. et al. (1990) Meth. Enzymol. 187:167).

Synthesis of 15(±)methyl-cyclo-LXA ⁴

[0160]

[0161] Compound o as the SiMe₃ derivative can be placed (˜1 gm) in around bottom 100 ml flask under an atmosphere enriched with argon indegassed benzene (20 ml). To this add 3.0 equivalents of a vinyl bromidefragment vide infra. This coupling reaction is carried out in catalyticamounts of Pd (PPh₃)₄ and Cul and can be monitored by injected aliquotsof this suspension into RP-HPLC monitored by UV abundance with a rapidscanning diode. The progression line course 1-3 h at 23° C. after whichthe material is extracted with ethyl acetate: H₂O 4:1 v/v) andconcentrated by rotoevaporation. The methyl ester can be saponified inLiOH/THF to give quantitative yields of the free carboxylic acid. Otherderivatives can be prepared as above using fragment A with differentfragment B moieties that have been substituted to give for example adimethyl or other derivative. This can be obtained by taking the readilyavailable ketone p and treating it with CH₃MgBr (60° C.) to generate qthat can also be coupled to fragment A as above using conventionaltechniques such as Pd(O)-Cu(I) coupling. Increased chain length fromC-15 can also be obtained.

Synthesis of 5-Methyl-LXB ⁴ and 4,4-Dimethyl-LXB ⁴

[0162] The 5-methyl -LXB₄ hinders or retards 5-oxo-LXB₄ formation. Usingthe general scheme outlined above, the A fragment can be constructed tocarry the 5-methyl in a vinyl bromide r precursor that is coupled to ajoined B+C fragment by Pd(0)-Cu(I) coupling.

[0163] The vinyl bromide r can be obtained from the s that containseither dimethyl or hydrogen substituents at its C-4 position. Theprotected precursor t containing fragments B+C is generated as reportedin reference (Nicolaou K. C. et al. (1991) Angew. Chem. Int. Ed. Engl.30:1100-16.). Compound t is converted to s or 28 by coupling with theindicated vinyl bromide. Thus the target molecule can be generated byadding r at 1.0 equv. (≈1 gm) to a round bottom flask degassedcontaining Et₂NH as solvent with t injected in Et₂NH at 1.2 equiv.Pd(Ph₃P)₄ is added at 0.02 equiv. to give the 8(9)-containing acetylenicprecursor methyl ester of s.

[0164] The material is extracted and subject to rotoevaporationsuspended in quinoline (0.5 eq) in CH₂Cl₂ and subject to hydrogenationusing (10%; 25° C.) Lindlar catalyst and a stream of H₂ gas toselectively reduce the acetylenic double bond at position 8. Theformation of the tetraene component of the methylester of 5-methyl-LXB₄or 4-dimethyl-LXB₄ methyl ester can be monitored by RP-HPLC to assesscompletion of the reduction (i.e., 1-3h). The methyl#esters are nextsaponified to their corresponding free acids by treating the productswith LiOH in THF 25 μl H₂O added at 0

24°, 8-24 h.

Example 4: Activity of Lipoxin Analogs on Columnar Epithelia

[0165] Several of the preferred lipoxin analogs (shown structurally ascompounds 1 through 8 in Example 3) were prepared by total synthesis asdescribed in Example 2. Following preparation and isolation of thesecompounds via HPLC, compounds were assessed to determine whether theyretain biological activity using the epithelial cell transmigrationassays as described above in Example 1.

[0166] Compounds 1 through 8 (10⁻⁷-10⁻¹⁰M) were found to inhibitneutrophil transmigration on epithelial cells. The acetylenic precursors( compound 1, 3, 5 and 7) were found to be physically more stable thantheir tetraene counterparts. Compound 7, which did not have an alcoholgroup in the C15 position or other modifications in the series, showedno biological activity in the assays. It would therefore appear that asubstituent in the C15 position of lipoxin is necessary for thebiological activity of at least lipoxin A₄ analogs. Lipoxin analogs 1through 8 were found to block migration at potencies greater than orequal to synthetic lipoxin A₄. Compounds 1, 2 and 4 were found to beparticularly effective. The results indicate that lipoxin A₄ analogswith modifications in C15-C20 positions retain their biological actionand can inhibit PMN transmigration in columnar epithelia.

Equivalents

[0167] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

We claim:
 1. A method for treating or preventing a disease or conditionassociated with columnar epithelial inflammation in a subject comprisingadministering to the subject an effective anti-inflammatory amount of apharmaceutical composition comprising a lipoxin A₄ compound and apharmaceutically acceptable carrier.
 2. A method of claim 1, wherein thelipoxin compound is an analog of natural lipoxin A₄.
 3. A method ofclaim 2, wherein the analog of natural lipoxin A₄ has a longer half-lifethan natural lipoxin A₄.
 4. A method of claim 3, wherein the lipoxinanalog is selected from the group consisting of:

where R′ is H or CH₃.
 5. A method of claim 1 wherein the columnarepithelium is an epithelium of the intestine, kidney, stomach, liver,thyroid, trachea, lung, gall bladder, urinary bladder, bile duct,pancreatic duct, or testicle.
 6. A method of claim 1 wherein the diseaseor condition is associated with inflammation of intestinal epithelia,selected from the group consisting of. acute self-limited enterocolitis;viral infections such as non-specific enteritis or specific viralenteritis; ulcerative colitis; Crohn's disease; diverticulitis;bacterial enterocolitis, such as salmonellosis, shigellosis,campylbacter enterocolitis, or yersinia enterocolitis; protozoaninfections such as amebiasis; helminthic infection; pseudomembraneouscolitis; duodenitis caused by infections, physical and chemicalinjuries, Celiac disease, allergic disease, immune disorders or stressulcers; lymphocytic colitis; collagenous colitis; diversion-relatedcolitis; acute self-limited colitis; microscopic colitis; solitaryrectal ulcer syndrome; Behcetf's disease; nonspecific ulcers of thecolon; secondary ulcers of the colon; ischemic bowel disease;vasculitis; peptic duodenitis; peptic ulcer; bypass enteritis;ulcerative jejunoileitis; nonspecific ulcers of the small intestine; andmalabsorptive disorders such as mucosal lesions associated with analtered immune response, with idiopathic AIDS enteropathy, with viral orbacterial infections, or with miscellaneous diseases such asmastocytosis or eosinophilic gastroenteritis, or is the result ofsurgery, allergy, chemical exposure, or physical injury.
 7. A method fortreating or preventing abnormal transportation of fluid, electrolytes,or nutrients by a columnar epithelium in a subject, comprisingadministering to the subject an effective anti-diuretic amount of apharmaceutical composition comprising a lipoxin A₄ compound, and apharmaceutically acceptable carrier.
 8. A method of claim 7, wherein thelipoxin compound is an analog of natural lipoxin A₄.
 9. A method ofclaim 8, wherein the analog of natural lipoxin A₄ has a longer half-lifethan natural lipoxin A₄.
 10. A method of claim 9, wherein the lipoxinanalog is selected from the group consisting of:

where R′ is Hor CH₃.
 11. A method of claim 7 wherein the colurnnarepithelium is an epithelium of the intestine, kidney, stomach, liver,thyroid, trachea, lung, gall bladder, urinary bladder, bile duct,pancreatic duct, or testicle.
 12. A method of screening for a compoundwhich inhibits the activation of an inflammatory cell which interactswith a columnar epithelium, comprising the steps of: i. pretreating theinflammatory cell with the compound; ii. placing pretreated inflammatorycells beside a prepared columnar epithelial barrier; and iii.Determining whether the compound inhibits the activation o aninflammatory cell which interacts with the epithelial barrier.
 13. Amethod of claim 12, wherein the activation is one or more actionsselected from the group consisting of: adhesion to the epithelium,migration across the epithelial barrier, release of bioactive molecules,or a combination thereof.
 14. A method of claim 13, wherein theinflammatory cell is selected from the group consisting of:monocyte/macrophage, eosinophil, T-lymphocyte, B-lymphocyte, naturalkiller cell, and polymorphonuclear leukocyte (PMN).
 15. A method ofclaim 14, wherein the epithelial barrier is formed from a cell lineselected from the group consisting of: Caco-2, IEC-6, T84, HT-29, MDCK,LLC-PK₁, and isolated alveolar epithelial cells grown in primaryculture.
 16. A method of claim 15, wherein the prepared columnarepithelial barrier is an intestinal epithelial barrier having achemotaciic agent on the opposite side of the barrier.
 17. A method ofclaim 16, wherein the prepared epithelial barrier has a permeableartificial membrane on one side to prevent membrane-membrane contactbetween the epithelial barrier and the inflammatory cell.
 18. A methodof claim 17, wherein the prepared epithelial barrier has cell-sizedobjects located in the interstitial spaces between the epithelialbarrier.
 19. A composition identified by the method of claim
 12. 20. Amethod of screening for a lipoxin compound that inhibitspolymorphonuclear leukocyte (PMN) adhesion to or migration across anepithelium, comprising the steps of: i. pretreating PMN cells with thelipoxin compound; ii. placing pretreated PMN on one side of a columnarepithelial carrier having a chemotactic agent on the other side; andiii. Determining whether the lipoxin or lipoxin compound modifies PMNadhesion to or migration across the columnar epithelial barrier.
 21. Amethod of claim 20, wherein the epithelial barrier is a monolayer formedfrom cells selected from the group consisting of: Caco-2, IEC-6, T84,HT-29, MDCK, LLC-PK₁, and isolated alveolar epithelial cells grown inprimary culture.
 22. A method of claim 21, wherein the epithelialbarrier is a monolayer of intestinal epithelial cells selected from thegroup of cell lines consisting of: T84, Caco-2, and IEC-6.
 23. A methodof claim 22, wherein the epithelial barrier is a monolayer of humanintestinal epithelial T84 cells.
 24. A method of claim 23, wherein thePMN chemotactic agent is selected from the group consisting of:leukotriene B₄, 12S-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid(12-HETE) , 5S-hydroxy-8, 11, 14-cis-6-trans- eicosatetraenoic acid(5-HETE), N-formyl-methionine-leucine-phenylalanine (fMLP), interleukin8 (IL-8), C5a, platelet activating factor (PAF), and TGF-β.
 25. A methodof claim 24, wherein the PMN chemotactic agent is fMLP.
 26. A method ofclaim 25, wherein the modification of PMN adhesion or migration ismeasured by a myeloperoxidase assay.
 27. A composition identified by themethod of claim
 20. 28. A method of screening for a compound whichreduces or eliminates the symptoms of secretory diarrhea caused byabnormal intestinal chloride ion secretion, comprising: i. combining aprepared epithelial barrier and inflammatory cells; ii. stimulatingchloride secretion by an intestinal epithelial barrier with an amount of5′-adenosine monophosphate (5′-AMP)or an agonist thereof; iii. exposingthe epithelial barrier to the compound; and iv. Determining whether thecompound affects chloride ion secretion to reduce or eliminate thesymptoms of secretory diarrhea.
 29. A method of claim 28, wherein thedetermination of the effectiveness of the compound is made by measuringthe electrical resistance of the epithelial barrier, the electricalresistance of the epithelial membrane, the endogenous current, or acombination thereof.
 30. A method of claim 29, wherein the 5′-AMPagonist is selected from the group consisting of: cyclic AMP, forskolin,and carbachol.
 31. A method of claim 30, wherein the compound is alipoxin analog, which optionally has a longer tissue half-life than thecorresponding lipoxin and optionally is actively absorbed by theintestine.
 32. A method of claim 31, wherein the epithelial barrier isformed from a cell line selected from the group consisting of: T84,Caco-2, and IEC-6.